Enzymatic Carbon−Carbon Bond Formation in Water-in-Oil Highly

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Langmuir 2003, 19, 1337-1346

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Enzymatic Carbon-Carbon Bond Formation in Water-in-Oil Highly Concentrated Emulsions (Gel Emulsions) Laia Espelt,†.‡ Pere Clape´s,‡ Jordi Esquena,† Albert Manich,§ and Conxita Solans*,† Department of Surfactant Technology, Department of Peptide and Protein Chemistry, and Department of Ecotechnologies, Institute for Chemical & Environmental ResearchsC.S.I.C., Jordi Girona 18-26, 08034-Barcelona, Spain Received September 25, 2002. In Final Form: November 1, 2002 Water-in-oil (W/O) highly concentrated emulsions (gel emulsions) of water/C14E4/aliphatic hydrocarbon systems were investigated as reaction media for the aldolic condensation of dihydroxyacetone phosphate (DHAP) with acceptor aldehydes such as phenylacetaldehyde (1) and benzyloxyacetaldehyde (2), catalyzed by D-fructose-1,6-bisphosphate aldolase from rabbit muscle (RAMA). Prior to any enzymatic reaction, both the formation and stability of the W/O gel emulsions in the presence of reactants were assessed. It was found that the aldehydes improved greatly the kinetic stability of W/O gel emulsions at 25 °C by decreasing the hydrophile-lipophile balance temperature (THLB) of the water/C14E4/aliphatic hydrocarbon systems. Interestingly, the stability of RAMA in W/O gel emulsions was improved by 7- and 25-fold compared to that in aqueous medium or conventional dimethylformamide/water 1/4 v/v mixture, respectively. It was found that the equilibrium yields and enzymatic activity depended on both the aldehyde partitioning between the continuous and dispersed phases and the water-oil interfacial tension. The highest enzymatic activities were achieved in W/O gel emulsion systems with the lowest water-oil interfacial tension. The equilibrium yield depended on the water-oil interfacial tension for the hydrophobic phenylacetaldehyde, and on the partition coefficient for the hydrophilic benzyloxyacetaldehyde. Optimum equilibrium product yields (65-70%) were achieved at either the lowest water-oil interfacial tension or partition coefficient values.

Introduction Colloidal surfactant systems (e.g., micelles, liquid crystals, microemulsions, vesicles, emulsions) are attracting a great deal of attention as alternative reaction media.1-5 Their advantages for this application are manifold. They possess micro- and nanostructures consisting of well-defined hydrophilic and lipophilic domains separated by surfactant films. As a result, their interfacial area is very large, the exchange between chemical species located in different domains is favored, and chemical reactions with higher reaction rates and yields can be achieved. Moreover, the self-assembled surfactant aggregates and the disperse phase of colloidal systems may act as micro- or nanoreactors where reactants are concentrated and consequently reaction yields can be considerably improved.1-3,6,7 These nanoreactors act also as templates allowing the preparation of nanoparticles and * To whom correspondence should be addressed: Telephone: 34-93-400 61 59. Fax: 34-93-204-59-04. E-mail: [email protected]. † Department of Surfactant Technology. ‡ Department of Peptide and Protein Chemistry. § Department of Ecotechnologies. (1) Kalyanasundaram, K.; Gra¨tzel, M. In Kinetics and Catalysis in Microheterogeneous Systems; Kalyanasundaram, K., Gra¨tzel, M., Eds.; Marcel Dekker: New York, 1991; pp 1-12. (2) Walde, P. Curr. Opin. Colloid Interface Sci. 1996, 1, 638-644. (3) Holmberg, K. In Industrial Applications of Microemulsions; Solans, C., Kunieda, H., Eds.; Marcel Dekker: New York, 1997; pp 6995. (4) Lo´pez-Quintela, M. A.; Quibe´n-Solla, J.; Rivas, J. In Industrial Applications of Microemulsions; Solans, C., Kunieda, H., Eds.; Marcel Dekker: New York, 1997; pp 247-265. (5) Antonietti, M. Curr. Opin. Colloid Interface Sci. 2001, 6, 244248. (6) Oberholzer, T.; Albrizio, M.; Luisi, P. L. Chem. Biol. 1995, 2, 677-682.

mesoporous materials with controlled size and shape.5,8-11 Furthermore, the use of colloidal systems enables total or partial replacement of organic solvents by aqueous media.12,13 In this work, attention has been focused on the study of highly concentrated emulsions as reaction media. These emulsions are also referred to in the literature as highinternal-phase-ratio emulsions (HIPRE)14-19 or gel emulsions.20-29 The term “gel emulsions” will be used through(7) Bunton, C. A.; Romsted, L. S. In Handbook of Microemulsion Science and Technology; Kumar, P., Mittal, K. L., Eds.; Marcel Dekker: New York, 1999; pp 457-482. (8) Attard, G. S.; Glyde, J. C.; Go¨ltner, C. G. Nature 1995, 378, 366368. (9) Schubert, K. V.; Lusvardi, K. M.; Kaler, E. W. Colloid Polym. Sci. 1996, 274, 875-883. (10) Pileni, M. P. Langmuir 1997, 13, 3266-3276. (11) Hubert, D. H. W.; Jung, M.; German, A. L. Adv. Mater. 2000, 12, 1291-1292. (12) Pinazo, A.; Infante, M. R.; Izquierdo, P.; Solans, C. J. Chem. Soc., Perkin Trans. 2 2000, 1535-1539. (13) Solans, C.; Pinazo, A.; Caldero, G.; Infante, M. R. Colloids Surf., A 2001, 176, 101-108. (14) Lissant, K. J. J. Colloid Interface Sci. 1966, 22, 462-468. (15) Princen, H. M. J. Colloid Interface Sci. 1979, 71, 55-66. (16) Lissant, K. J.; Mayhan, K. G. J. Colloid Interface Sci. 1973, 42, 201-208. (17) Princen, H. M. J. Colloid Interface Sci. 1983, 91, 160-175. (18) Princen, H. M.; Kiss, A. D. J. Colloid Interface Sci. 1986, 112, 427-437. (19) Princen, H. M.; Aronson, M. P.; Moser, J. C. J. Colloid Interface Sci. 1980, 75, 246-270. (20) Solans, C.; Comelles, F.; Azemar, N.; Sanchez-Leal, J.; Parra, J. L. J. Com. Esp. Deterg. 1986, 17, 109-122. (21) Kunieda, H.; Solans, C.; Shida, N.; Parra, J. L. Colloids Surf., A 1987, 24, 225-237. (22) Solans, C.; Dominguez, J. J. G.; Parra, J. L.; Heuser, J.; Friberg, S. E. Colloid Polym. Sci. 1988, 266, 570-574. (23) Solans, C.; Azemar, N.; Parra, J. L. Prog. Colloid Polym. Sci. 1988, 76, 224-227.

10.1021/la020811b CCC: $25.00 © 2003 American Chemical Society Published on Web 01/25/2003

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Figure 1. Micrograph of a W/O gel emulsion viewed under normal light.

out this work. This type of emulsions is characterized by volume fractions of dispersed phase higher than 0.73, the critical value of close-packed monodispersed spheres.14,15,30 Consequently, the droplets are deformed and/or polydisperse, separated by a thin film of continuous phase (Figure 1). This foamlike structure confers on them a viscoelastic rheological behavior responsible for their gel appearance.22,31 All these properties make them of particular interest for theoretical studies and for specific applications.32-36 One of the remarkable applications of gel emulsions is their use as novel reaction media. In this context, polymerization reactions in the continuous and/or dispersed phases have been reported long ago.32,33,35,36 More recently, chemical synthesis of a cationic surfactant by a condensation reaction between a fatty acid and an amino acid12,13 as well as enzymatic peptide synthesis using R-chymotrypsin as catalyst37 have been reported using water-in-oil (W/O) gel emulsions, formulated with 90 wt % water, as reaction media. The encouraging results of these studies have prompted us to further investigate the use of W/O gel emulsions as effective and environmentally friendly reaction media for enzymatic biotransformations. (24) Kunieda, H.; Yano, N.; Solans, C. Colloids Surf., A 1989, 36, 313-322. (25) Kunieda, H.; Evans, D. F.; Solans, C.; Yoshida, M. Colloids Surf., A 1990, 47, 35-43. (26) Solans, C.; Pons, R.; Zhu, S.; Davis, H. T.; Evans, D. F.; Nakamura, K.; Kunieda, H. Langmuir 1993, 9, 1479-1482. (27) Pons, R.; Ravey, J. C.; Sauvage, S.; Stebe, M. J.; Erra, P.; Solans, C. Colloids Surf., A 1993, 76, 171-177. (28) Pons, R.; Erra, P.; Solans, C.; Ravey, J. C.; Stebe, M. J. J. Phys. Chem. 1993, 97, 12320-12324. (29) Kunieda, H.; Rajagopalan, V.; Kimura, E.; Solans, C. Langmuir 1994, 10, 2570-2577. (30) Ostwald, W. Wilmersdorf. Z. Chem. Ind. Kolloide 1910, 8, 103109. (31) Solans, C.; Pons, R.; Kunieda, H. Gel emulsionssrelationship between phase behavior and formation. In Modern Aspects of Emulsion Science; Binks, B. P., Ed.; The Royal Society of Chemistry: Cambridge, UK, 1998; pp 367-394. (32) Bampfield, H. A.; Cooper, J. In Encyclopedia of Emulsion Technology; Becher, P., Ed.; Marcel Dekker: New York; Vol. 3; pp 281306. (33) Williams, J. N.; Gray, A. J.; Wilkerson, M. H. Langmuir 1990, 6, 437-444. (34) Bibette, J.; Roux, D.; Nallet, F. Phys. Rev. Lett. 1990, 65, 24702473. (35) Cameron, N. R.; Sherrington, D. C. Adv. Polym. Sci. 1996, 126, 163-214. (36) Ruckenstein, E. Adv. Polym. Sci. 1997, 127, 1-58. (37) Clape´s, P.; Espelt, L.; Navarro, M. A.; Solans, C. J. Chem. Soc., Perkin Trans. 2 2001, 1394-1399.

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Herein, we investigated the application of W/O gel emulsions as reaction media for carbon-carbon bond formation catalyzed by rabbit muscle D-fructose-1,6bisphosphate aldolase (RAMA). The aldol condensation of dihydroxyacetone phosphate (DHAP) with phenylacetaldehyde (1) or benzyloxyacetaldehyde (2) was used as model reaction (Scheme 1). The stereoselective carboncarbon bond formation catalyzed by dihydroxyacetone phosphate (DHAP) dependent aldolases has focused a tremendous interest in recent years, especially in the asymmetric synthesis of carbohydrates and complex carbohydrate-like molecules.38-42 DHAP aldolases catalyze the aldol addition of the highly specific DHAP donor substrate with a variety of aldehyde acceptors. In many instances the solubility characteristics of both donor (DHAP) and acceptor (aldehyde) substrates differ substantially. While DHAP is fully soluble in aqueous media, and insoluble in organic solvents including the most polar ethanol or methanol, the solubility of the acceptor is generally reverse. Aqueous-organic cosolvent mixtures, namely dimethylformamide/water mixtures, are normally used to overcome this problem. Nevertheless, 10%-20% v/v cosolvent concentration is usually not enough for substrate solubility and is often detrimental to the particularly sensitive aldolase.43-45 Aware of these limitations, and with the aim to contribute to a better knowledge of the role of gel emulsions as reaction media for enzymatic biotransformations, the objective of this work was to investigate the most critical reaction variables affecting the enzymatic activity and product yield in W/O gel emulsions formulated with high water content (i.e., 90 wt %). Experimental Section Materials. Octane, decane, dodecane, tetradecane, and hexadecane were obtained from Sigma (St. Louis, MO). Technical grade poly(oxyethylene) tetradecyl ether surfactant, with an average of 4 mol of ethylene oxide per surfactant molecule, abbreviated as C14E4, was obtained from Albright & Wilson (Barcelona, Spain). Fructose-1,6-bisphosphate aldolase from rabbit muscle (RAMA), (EC 4.1.2.13, powder white, 15-20 U/mg) was from Fluka (Buchs, Switzerland). Acetonitrile HPLC isocratic grade was from Merck (Darmstadt, Germany). Dihydroxyacetone phosphate dimer bis(ethyl ketal) was from Sigma. Phenylacetaldehyde and benzyloxyacetaldehyde were from Aldrich (Milwaukee, WI). Deionized water was used for preparative HPLC, and MilliQ grade water was used for both analytical HPLC and gel emulsion formation. Methods. HLB temperature (THLB) was determined by measuring the conductivity of emulsions as a function of temperature. A Crison conductivity meter Model 525 (Barcelona, Spain), with parallel Pt/platinized electrodes with 0.998 cm-1 cell, was used. The cell constant was determined using standard KCl solutions. Aqueous NaCl solution, 10-2 M, was used for electrical conduction instead of water. Each curve was repeated three times, and the standard error of the calculated HLB temperature ranged from 0.4-1.3 °C. (38) Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T. Angew. Chem., Int. Ed. Engl. 1995, 34, 2-432. (39) Fessner, W.-D.; Walter, C. Top. Curr. Chem. 1997, 184, 97-194. (40) Machajewski, T. D.; Wong, C. H. Angew. Chem., Int. Ed. 2000, 39, 1353-1374. (41) Roberts, S. M. J. Chem. Soc., Perkin Trans. 1 2001, 1475-1499. (42) Wong, C.-H.; Whitesides, G. M. Enzymes in Synthetic Organic Chemistry, 1st ed.; Elsevier Science Ltd.: Kidlington, Oxford, UK, 1994; Vol. 12. (43) Bednarski, M. D.; Simon, E. S.; Bischofberger, N.; Fessner, W. D.; Kim, M. J.; Lees, W.; Saito, T.; Waldmann, H.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 627-635. (44) Budde, C. L.; Khmelnitsky, Y. L. Biotechnol. Lett. 1999, 21, 7780. (45) Sobolov, S. B.; Bartoszko-Malik, A.; Oeschger, T. R.; Montelbano, M. M. Tetrahedron Lett. 1994, 35, 7751-7754.

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Scheme 1. Reversible Aldol Reaction between the Donor Dihydroxyacetone Phosphate (DHAP) and the Acceptor Aldehydes Phenylacetaldehyde (1) and Benzyloxyacetaldehyde (2), Catalyzed by D-Fructose-1,6-Bisphosphate Aldolase from Rabbit Muscle (RAMA)

Interfacial tension was measured by the spinning drop technique using a Kru¨ss Site 04 tensiometer (Hamburg, Germany), at constant temperature. The dispersed and continuous phases were obtained from equilibrated samples at constant temperature. Each experiment was done in triplicate, and the estimated standard errors are given in Table 4. Density was measured with an AP, DMA46 thermostated density meter (Graz, Austria). Partition Coefficients of the Aldehydes. The aldehydes (0.06 mmol) were added to an aqueous organic two-phase system consisting of MilliQ water (1 mL) and the corresponding oil (1 mL) in a screw-capped 4 mL flat-bottom vials. The vials were placed on a thermostated reciprocal shaker (150 rpm) at 25 °C for 6-7 h. Then samples (50 µL) were withdrawn from both phases, dissolved with MeOH (1 mL), and analyzed by HPLC. Each experiment was done in triplicate, and the estimated standard errors are given in Table 1. 1H and 13C NMR spectra were recorded with a Unity-300 spectrometer from Varian (Palo Alto, CA) for D2O or d6-MeOH solutions. Preparation of Gel Emulsions. Gel emulsions were prepared by slow addition of the internal or dispersed phase (water or an aqueous solution) to a homogeneous mixture of the external or continuous phase (surfactant, oil, and oil-soluble reactants) while stirring continuously with a vortex mixer. All emulsions were prepared under the same rates of addition and stirring to obtain reproducible emulsions. Enzymatic Reactions in Gel Emulsions. Reactions were carried out in screw-capped 10 mL round-bottom test tubes. The aldehyde (0.125 mmol, 50 mM based on the reaction volume), the oil (0.15 g), and the surfactant (0.1 g) were mixed vigorously. To this mixture, the DHAP solution (2.25 mL, 0.075 mmol, 30 mM), freshly prepared and adjusted to pH 6.95 with NaOH as described by Effenberger et al.,46 was added dropwise while stirring at 25 °C. Finally, RAMA (100 µL of a 20 mg/mL solution giving 2 mg of enzyme) was added to the gel emulsion under stirring. Then the reactors were placed on a thermostated bath at 25 °C without agitation, unless otherwise stated. The enzymatic synthesis of the products (3S,4R)-1,3,4-trihydroxy-5-phenyl-2-pentanone 1-phosphate (3) and 5-O-benzyl-D-xylulose 1-phosphate (4) was performed at milligram scale for their characterization by NMR spectroscopy. To this end, the enzymatic reactions in gel emulsions were scaled up at high substrate concentration using the experimental procedure described above, with the following amounts of reactants and enzyme: aldehyde (0.625 mmol), DHAP solution (4.5 mL, 0.45 mmol), and RAMA (200 µL of a solution 20 mg/mL, giving 4 mg of enzyme); 75-90 mg (50-55% isolated yields based on DHAP) of pure product were obtained. The 1H and 13C NMR spectra of products 3 and 4 were consistent with the reported values.43,47 Sampling and HPLC Analysis. Samples (50 mg) were withdrawn from the reaction medium at 30 min, 2 h, 4 h, 7 h, and 24 h, dissolved with methanol to stop any enzymatic reaction, and analyzed subsequently by HPLC. HPLC analyses were performed on a Merck-Hitachi Lichrograph system (Darmstadt, Germany) using a Lichrocart HPLC cartridge, 250 × 4 mm filled (46) Effenberger, F.; Straub, A. Tetrahedron Lett. 1987, 28, 16411644. (47) Humphrey, A. J.; Turner, N. J.; McCague, R.; Taylor, S. J. C. J. Chem. Soc., Chem. Commun. 1995, 2475-2476.

with Lichrosphere 100, RP-18, 5 µm, from Merck. The solvent system was the following: solvent A, 0.10% v/v trifluoroacetic acid (TFA) in H2O; solvent B, 0.09% v/v TFA in H2O/CH3CN 1:4; flow rate 1 mL/min, detection at 254 nm. The elution condition for the separation of the components in each synthesis was a gradient from 10% B to 70% B over 30 min; retention factors (k′) for aldehydes and products were 17.7 for 1, 7.7 for 3, 14.2 for 2, and 9.7 for 4. Quantitative analysis of products was performed from peak areas by the external standard method. The initial reaction rates of product formation (v° µmol/min‚mg of enzyme) were calculated from the time-progress curves. Quantitative analysis of DHAP was performed using an enzymatic assay.48 In all cases, experiments were performed in triplicate and the standard error was estimated in each case. Stability of RAMA. The reaction media (1 g of W/O gel emulsion, 1 mL of pure buffer, or 1 mL of DMF/water 1/4 v/v) containing the enzyme (2 mg for each medium, giving a final concentration of 2 mg/g of emulsion or mL) and phenylacetaldehyde (50 mM) were incubated at 25 °C. One reactor for each required incubation time was used. The activity of the enzyme was tested by adding to the reaction media a solution of DHAP, prepared as indicated above, in W/O gel emulsion (1 g, 0.9 g of 30 mM aqueous DHAP solution, 0.04 g of C14E4, and 0.06 g of aliphatic hydrocarbon) or in solution (0.9 mL, 30 mM DHAP), measuring the condensation product 3 formed after 1 and 2 h by HPLC. In all cases, experiments were performed in triplicate. Unit Definition: One unit (U) corresponds to the amount of enzyme that converted 1.0 µmol of DHAP to (3S,4R)-1,3,4trihydroxy-5-phenyl-2-pentanone 1-phosphate 3 per minute at pH 7 and 25 °C.

Results and Discussion Emulsions of the ternary water/C14E4/aliphatic hydrocarbon systems were chosen as reaction media, as it was shown previously12,13,37 that stable W/O gel emulsions, with a variety of additives, were produced in these systems. In ternary water/poly(ethylene oxide)-type nonionic surfactant/aliphatic hydrocarbon systems, W/O gel emulsions form at temperatures above the hydrophile-lipophile balance (HLB) temperature (THLB) of the corresponding system.21,24,29 THLB is the temperature at which the nonionic surfactants change their preferential solubility from water to oil or vice versa. It should be noted that the nonionic surfactant used in this work was of technical grade; consequently, the THLB is not a system property but depends on composition due to the different distribution of homologues.49 It has been shown that stability of W/O gel emulsions increases with temperature reaching a maximum at 25-30 °C above the THLB.21,24-26 In addition to temperature, other factors such as the oil, the structure of the continuous phase, and the presence of additives (48) Bergmeyer, H. U. Methods of Enzymatic Analysis, 3rd ed.; Bergmeyer, J., Grassl, M., Eds.; Verlag Chemie: Deerfield Beach, FL, 1983; Vol. 2, pp 146-147. (49) Kunieda, H.; Ishikawa, N. J. Colloid Interface Sci. 1985, 107, 122-128.

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Figure 2. Conductivity as a function of temperature in the ternary water/C14E4/hexadecane system with 4 wt % surfactant concentration and equal water/hexadecane weight ratio, without aldehyde (2) and in the presence of aldehydes (50 mM) 1 (9) and 2 (b).

have a drastic influence on gel emulsion formation and stability.26,29,50,51 W/O gel emulsions formulated with high water content (90 wt %) stable at the reaction temperature, 25 °C, were selected for the aldolase-catalyzed carbon-carbon bond formation reactions. This reaction temperature was chosen because it is the most commonly employed in these reactions. The effect of the substrates on gel emulsion properties (e.g., stability) as well as the stability of the enzyme in the emulsions was assessed prior to the reactivity tests. Gel Emulsion Formation and Stability. Incorporation of the reaction components into the gel emulsions revealed that neither the donor substrate DHAP nor the enzyme RAMA, at the concentrations used for carrying out the reactions (30 mM DHAP and 0.8 mg RAMA/g of emulsion, respectively), produced any change in the macroscopic properties of the emulsions (droplet size and stability). The droplet size of the emulsions ranged from 0.8 to 2 µm diameter as detected by optical microscopy. In contrast, addition of 50 mM of either phenylacetaldehyde (1) or benzyloxyacetaldehyde (2) increased gel emulsion stability and decreased the THLB of the system. No macroscopic changes were observed after 24 h as revealed by optical microscopy. An example of the effect of the aldehyde acceptors 1 and 2 on the THLB is shown in Figure 2, where conductivity is plotted as a function of temperature for the water/C14E4/ hexadecane system (water/oil weight ratio equal to 1 and 4 wt % C14E4 concentration), without and with a constant concentration of each aldehyde (50 mM). The results depicted in Figure 2 show that the shape of the conductivity-temperature curves was similar for the system without or with aldehyde. However, the pronounced decrease in conductivity to values close to zero, indicating transition of the emulsion from O/W to W/O (i.e., the THLB), (50) Pons, R.; Carrera, I.; Erra, P.; Kunieda, H.; Solans, C. Colloids Surf., A 1994, 91, 259-266. (51) Kunieda, H.; Fukui, Y.; Uchiyama, H.; Solans, C. Langmuir 1996, 12, 2136-2140.

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Figure 3. Influence of aldehydes 1 (9) and 2 (b) concentrations on the THLB of the same system and composition as that in Figure 2.

was different for the three systems. Without aldehyde, the THLB is 58.5 ( 0.4 °C. The addition of aldehyde produced a shift in the transition temperature to lower temperatures, the effect being larger for phenylacetaldehyde (1) (THLB ) 47.5 ( 0.5 °C) than for benzyloxyacetaldehyde (2) (THLB ) 53.8 ( 1.3 °C). The effect of aldehyde concentration on the THLB was also studied using the same emulsion system as in the above example. As shown in Figure 3, the higher the aldehyde concentration, the lower the THLB. Considering the chemical structure of these aldehydes (see Scheme 1), the effect of decreasing the THLB should be expected. It is known that poly(oxyethylene) chains are highly soluble in aromatic hydrocarbons.52,53 Therefore, aromatic rings may penetrate the surfactant layers, making them fragile.24 This penetration causes a decrease in the hydrogen bonding between the surfactant polar head and water molecules, rendering the surfactant more lipophilic (i.e., decreasing THLB). It has also been reported that the addition of a polar oil into a system with a nonpolar or less polar oil produces a reduction in the HLB temperature: the more polar (less hydrophobic) the added oil, the higher is the reduction.54 To gain a better understanding of the influence of the aldehydes on the THLB of the system, their partition coefficient (P) between the oil (i.e., octane, decane, and hexadecane, used in this work) and water was experimentally measured. The results, presented in Table 1, showed that aldehyde 1 mainly partitioned in the oil phase while aldehyde 2 partitioned preferentially in the aqueous phase. In both cases, the partition decreased moderately with the increase in the hydrocarbon chain length. Aldehyde 1 seems to play the role of a cosolvent, influencing more drastically the interfacial properties than aldehyde 2 and, therefore, its ability to reduce more strongly the HLB temperature of the system. (52) Christenson, H.; Friberg, S. E. J. Colloid Interface Sci. 1980, 75, 276-285. (53) Bailey, F. E.; Koleske, J. V. In Nonionic surfactants. Physical Chemistry; Schick, M. J., Ed.; Marcel Dekker: New York, 1987; pp 927-969. (54) Kunieda, H.; Horii, M.; Koyama, M.; Sakamoto, K. J. Colloid Interface Sci. 2001, 236, 78-84.

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Table 1. Partition of Phenylacetaldehyde (1) and Benzyloxyacetaldehyde (2) in Water-Oil Two-Phase Systems partition coefficients (o/w) oil

1

2

octane decane dodecane tetradecane hexadecane

2.83 ( 0.13 2.96 ( 0.10 2.78 ( 0.16 2.55 ( 0.09 2.47 ( 0.06

0.36 ( 0.03 0.27 ( 0.01 0.26 ( 0.02 0.22 ( 0.01 0.21 ( 0.01

In the water/C14E4/hexadecane system, gel emulsions with 90 wt % water concentration could not be formed at 25 °C and those of the corresponding system with tetradecane were highly unstable at this temperature. Addition of 50 mM of either phenylacetaldehyde 1 or (benzyloxy)acetaldehyde 2 allowed the preparation of stable gel emulsions, although those formulated with hexadecane and 2 became fluid within a few hours after preparation. These results can be explained considering the relationship between THLB and stability of gel emulsions. The THLB of the emulsions with 90 wt % aqueous component could not be determined experimentally by conductivity measurements due to the instability of the corresponding emulsions near the THLB. However, the values were extrapolated from the plots of Figure 4, which shows the variation of THLB with water concentration in the water/C14E4/hexadecane (Figure 4a) and water/C14E4/ tetradecane (Figure 4b) systems without and with a constant concentration of aldehyde (50 mM). The hydrocarbon/C14E4 (O/S) weight ratio was 60/40. The plots of Figure 4 are consistent with the predicted linear relationship between THLB and concentration.49 Extrapolation of the curves to 90 wt % water gave the estimated THLB values (Table 2). The results of gel emulsion formation and stability, described above, are readily explained by the data from Table 2. By addition of aldehydes 1 and 2 to the hexadecane system, THLB decreased from 25.4 °C, without aldehyde, to 18.1 and 21.6 °C, respectively; thus W/O emulsions could be obtained at 25 °C with the addition of aldehyde to the system. It is well-known that the closer the experimental temperature to the THLB the more unstable the gel emulsions are.21-26,31 Consequently, at 25 °C, the W/O gel emulsions formulated with the aldehydes would have higher stability than those without these substrates. Enzyme Stability in Gel Emulsions. Once the formation and stability of the W/O gel emulsions in the presence of the reaction components was established, the catalytic stability of the enzyme in these systems was also evaluated. Enzyme stability is a crucial issue in biocatalysis. Indeed, an appropriate reaction medium must offer, among other things, a good stability to the enzyme. Thus, the stability of RAMA in a W/O gel emulsion with 90 wt % water and C14E4/oil weight ratio of 40/60 containing 50 mM aldehyde 1 at 25 °C was measured and compared to that in aqueous buffer and in conventional dimethylformamide (DMF)/aqueous buffer 1/4 v/v medium. Octane, dodecane, and hexadecane were the oil components selected for the gel emulsions. No phase separation was observed during the 18 days of incubation. The results, depicted in Figure 5, showed a highly improved stability of the enzyme in the W/O gel emulsion system with hexadecane as compared to that in the two other media. The half-life of the enzymatic activity in gel emulsion was 100 ( 10 h, while in aqueous and in DMF/ water 1/4 v/v media it was only 14 ( 4 and 4 ( 2 h, respectively. As reported by other authors,44,45 the presence of 20% v/v DMF had a dramatic effect on the stability of

Figure 4. THLB as a function of water concentration in ternary water/C14E4/oil systems with constant C14E4 concentration (4 wt %). (a) Water/C14E4/hexadecane system without (2) and with 50 mM aldehydes 1 (9) and 2 (b). (b) Water/C14E4/tetradecane system without (2) and with 50 mM aldehyde 2 (b). Table 2. THLB of Water/C14E4/Oil System with 90 wt % Water and C14E4/Oil 40/60 wt % without and with a Constant Concentration of Phenylacetaldehyde 1 and Benzyloxyacetaldehyde 2 (50 mM)a THLB system

no aldehyde

aldehyde 1

aldehyde 2

W/C14E4/tetradecane W/C14E4/hexadecane

19.3 ( 1.3 25.4 ( 0.5

ndb 18.1 ( 1.1

17.6 ( 2.2 21.6 ( 0.8

a The T HLB values were extrapolated from the plots of Figure 4. The standard errors were estimated from the statistics of the linear regression curves of Figure 4. b nd, not determined.

RAMA. It is also noteworthy that the enzyme stability was independent of the oil used to formulate the W/O gel emulsions (results not shown). Enzymatic Synthesis in W/O Gel Emulsions. It is important to point out that the dihydroxyacetone phos-

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Figure 6. Schematic representation of the reaction medium indicating the most likely location of the components for aldolase-catalyzed carbon-carbon bond formation in W/O gel emulsions. Figure 5. Stability of rabbit muscle aldolase (RAMA) in a W/O gel emulsion of the water/C14E4/hexadecane system with 90 wt % water and hexadecane/C14E4 weight ratio 60/40 (3), dimethylformamide/water 1/4 v/v mixture (×), and pure aqueous buffer media (]). In all instances, the concentration of the phenylacetaldehyde was 50 mM. Experimental details are given in the Methods section.

phate (DHAP) substrate is not stable in solution, particularly at alkaline pH values, and decomposes into methylglyoxal.39 We have tested the stability of the DHAP at pH 6.95 (i.e., the reaction pH), in W/O gel emulsion and aqueous media. It was found that in all media the DHAP was stable within 4-6 h, and that 40% of the initial concentration was degraded after 24 h. Hence, the maximum product yields (i.e., equilibrium yields) of the reaction should be reached between 2 and 6 h. For longer incubation times the decomposition of DHAP may have to be considered because this affects the equilibrium position of the reaction. In the present study, the amount of enzyme used was adjusted to obtain reaction rates fast enough to reach a constant product concentration in 2-4 h. Under the reaction conditions used, we observed that the maximum product concentration was achieved around 4 h, remaining invariable up to 7 h and, in most instances, up to 24 h. Since the enzyme was fully active over this period (see Figure 5), the concentrations achieved within 4-6 h were considered close to the equilibrium. The aldolic additions of DHAP with the acceptor aldehydes 1 and 2 catalyzed by RAMA were carried out at 25 °C using gel emulsions with similar THLB values to facilitate the interpretation of the results. Thus, W/O gel emulsions with 90 wt % water and oil/surfactant weight ratio of 60/40 belonging to the systems water/C14E4/ hexadecane for phenylacetaldehyde (1) (THLB ) 18.1 ( 1.1 °C) and water/C14E4/tetradecane for benzyloxyacetaldehyde (2) (THLB ) 17.6 ( 2.2 °C) were selected. In all cases, the aldehyde concentration was 50 mM. The gel emulsion structure was retained during the enzymatic reaction. No increase in drop size was observed in 24 h, as revealed by optical microscopy. The equilibrium yields found for (3S,4R)-1,3,4-trihydroxy-5-phenyl-2-pentanone 1-phosphate (3) and 5-Obenzyl-D-xylulose 1-phosphate (4) were 63 ( 3% and 64 ( 4%, respectively. These results demonstrated that it was possible to perform aldolic additions catalyzed by

RAMA in W/O gel emulsions, with yields comparable to those obtained in a conventional DMF/water 1/4 v/v mixture (70 ( 5%). The purification of 3 and 4 was carried out as in conventional DMF/water 1/4 system. Hence, anion exchange chromatography was also employed, taking advantage of the phosphate group present in the molecules. Thus, once the crude reaction mixture was loaded onto the column, the neutral, uncharged, or cationic material was washed away by EtOH/water mixtures. Then, the product was eluted with gradients of NaCl in 60/40EtOH/water. This methodology gave satisfactory results for both reaction media. Encouraged by these results, our next interest was to identify the critical parameters that control both the enzymatic activity and the product yield in W/O gel emulsion. To throw light on this point, some features about the reaction system were considered first. It can be assumed that the water-soluble components of the reaction, i.e., DHAP and the enzyme, are mainly located in the dispersed (aqueous) phase while the aldehydes partition between the continuous (oil), interface, and aqueous phases (Figure 6). The products 3 and 4 are highly watersoluble and will be mainly located in the aqueous phase. Although the precise mechanisms of the reactions in W/O gel emulsion media are not known yet, it is likely that the reaction takes place either at the oil/water interface55 or in the dispersed phase that contains the enzyme and the donor DHAP. Thus, the equilibrium position of the reaction and the enzymatic activity will depend on the relative changes in partitioning and/or solvation of the acceptor aldehydes in the continuous (i.e., oil phase) and dispersed (i.e., water) phases.56,57 Furthermore, although the interfaces can usually be neglected in thermodynamic analysis, in these systems they may influence significantly the equilibrium position.57 This is due to that a significant proportion of the substrate molecules present, mostly the acceptor aldehydes, may be located at the interfacial regions. The interfacial area per unit volume of the W/O gel emulsions is quite large: it can be estimated to be around 3.4 m2/mL. The substrate partitioning and/or solvation affects also both the kinetic behavior and the enzymatic activity: the (55) Hickel, A.; Radke, C. J.; Blanch, H. W. Biotechnol. Bioeng. 1999, 65, 425-436. (56) Halling, P. J. Biotechnol. Bioeng. 1990, 35, 691-701. (57) Halling, P. J. Enzyme Microb. Technol. 1994, 16, 178-206.

Carbon-Carbon Bond Formation in W/O Emulsions

aldehyde concentration seen by the biocatalyst will depend on its partition in the aqueous phase, that is, on its relative solvation in the nonpolar phase. Consequently, both the initial reaction rate (i.e., the enzymatic activity) and the apparent Km values will be affected by these parameters. The accessibility of the reactants to enzyme will also depend on their ability to cross the interface easily and, therefore, on the interfacial properties.58,59 Moreover, if the acceptor substrate is mainly located at the interface, the interfacial properties can modify its solvation in this region affecting the enzymatic activity as well. Since the oil/water interfacial tension changes dramatically and reaches a minimum at the THLB, one way to study the effect of the interfacial properties on the reaction performance is to measure the enzyme activity and product equilibrium yields at temperatures close to the THLB. Effect of Temperature. The initial reaction rate (v°) and product yields at equilibrium were measured as a function of temperature in a range between 18 and 30 °C. The systems selected were water/C14E4/hexadecane and water/C14E4/tetradecane for the reaction with phenylacetaldehyde and benzyloxyacetaldehyde, respectively (see above). Both systems gave W/O gel emulsions at this temperature range. However, at temperatures close to the THLB of the system, the kinetic stability of the W/O gel emulsions is low.24 The gel emulsions with benzyloxyacetaldehyde (2) experienced phase separation at 18 °C during the reaction. Therefore, all the assays were performed under stirring (horizontal shake, 150 rpm) to ensure homogenization of the reaction mixture. For the sake of comparison, the reactions were also conducted in DMF/aqueous buffer 1/4 v/v mixtures. Figure 7 depicts the initial reaction rate (v°) plotted against temperature in an Arrhenius type plot for the reaction in W/O gel emulsion and in DMF/aqueous buffer mixture. Overall, the reaction rates in DMF/water systems were slightly higher than that in W/O gel emulsion. Interestingly, the linear plots for both reaction systems had similar slopes. This means that the activation energy of the enzymatic reaction in DMF/water 1/4 v/v mixture and in W/O gel emulsions was the same. These results suggest that diffusional limitations in the W/O gel emulsions might be neglected,37 as well as changes in the enzyme conformation induced by the variations in the water-oil interfacial tension.60,61 It is noteworthy that similar initial reaction rates were obtained for the reactions with both phenylacetaldehyde (1) and benzyloxyacetaldehyde (2) in the temperature range studied. The effect of temperature on the product yields at equilibrium is depicted in Figure 8. As can be seen, using W/O gel emulsions there was no influence of the temperature on the equilibrium yields within 18 and 30 °C. In DMF/water mixtures a maximum product yield was observed at 25 °C, decreasing at 30 °C. We have not studied the reaction performance in DMF/water systems in detail, but a visual inspection of the reaction mixture at 30 °C revealed the presence of a precipitate after 1 h of reaction, which was not noticed at lower temperatures. This was probably due to, among other things, a fast and irreversible deactivation of the aldolase.44 In contrast, the enzyme in the W/O gel emulsions was fully active over a period of 24 h in the temperature range studied. (58) Gan, Q.; Baykara, F.; Rahmat, H.; Weatherley, L. R. Catal. Today 2000, 56, 179-190. (59) Paiva, A. L.; Balcao, V. M.; Malcata, F. X. Enzyme Microb. Technol. 2000, 27, 187-204. (60) Fink, A. L. Biochemistry 1973, 12, 1736-1742. (61) Fink, A. L.; Angelides, K. L. Biochemistry 1976, 15, 5287-5293.

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Figure 7. Arrhenius plots of v° for the aldolase-catalyzed synthesis of 3 in W/O gel emulsions of the water/C14E4/oil system with 90 wt % water and oil/C14E4 weight ratio 60/40 (0) and in DMF/water 1/4 v/v media (O). Conditions were aldehydes (50 mM), DHAP (30 mM), RAMA (0.8 mg/g or mL of reaction medium). Similar results were obtained for the synthesis of 4 (data not shown). The water/C14E4/hexadecane system (2.5 g) was used for the reaction with aldehyde 1 (synthesis of 3) and the water/C14E4/tetradecane system (2.5 g) for that with 2 (synthesis of 4). The final volume for the reactions carried out with DMF/water mixtures was 2.5 mL.

Figure 8. Influence of the reaction temperature on the product yield for the aldolase-catalyzed synthesis of 3 in W/O gel emulsions of the water/C14E4/oil systems (same composition as in Figure 7) (9) and in DMF/buffer 1/4 v/v media (0). Similar results were obtained for the synthesis of 4 (data not shown). The reaction conditions were aldehydes (50 mM), DHAP (30 mM), RAMA (0.8 mg/g or mL of reaction medium). The water/ C14E4/hexadecane system (2.5 g) was used for the reaction with aldehyde 1 (synthesis of 3) and the water/C14E4/tetradecane system (2.5 g) for that with 2 (synthesis of 4).

Considering the THLB of the W/O gel emulsions assayed, the water-oil interfacial tension at 18 °C was minimum, and the measured value for 30 °C was 0.098 ( 0.006 mN/m

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Table 3. Theoretical Hydrophile-Lipophile Balance (HLB) Temperaturea for the Ternary Water/C14E4/Oil System oil

THLB, °C

octane decane dodecane tetradecane hexadecane

4 9 14 20 22

a Calculated according to the equation reported by Kunieda et al.49

Table 4. Experimental Values of Water-Oil Interfacial Tension (γW/O) at 25 °C as a Function of the Oil Component for the Ternary Water/C14E4/Oil System with 90 wt % Water, C14E4/Oil Weight Ratio of 40/60, and with Constant Concentration of Phenylacetaldehyde (50 mM) oil

γ, mN/m

octane decane dodecane tetradecane hexadecane

0.296 ( 0.008 0.237 ( 0.009 0.150 ( 0.010 0.086 ( 0.009 0.037 ( 0.007

in the system with hexadecane and aldehyde 1. This suggests that, within this temperature range, the initial reaction rate appeared to be influenced solely by the temperature rather than by the water-oil interfacial tension of the system. Higher values of the water-oil interfacial tension can be achieved by raising the reaction temperature further above 30 °C. However, at those temperatures the stability of both RAMA and the donor substrate, DHAP, decreased considerably. Another reaction variable to consider is the type of oil. The oil can affect both the interfacial properties of the system and the substrate partition between the continuous and dispersed phases. Influence of the Oil Alkyl Chain Length. The changes in the interfacial properties produced by varying the temperature in a given system can also be afforded at constant temperature by changing a system component, e.g., the oil. Table 3 shows the THLB, calculated from Kunieda’s equation,62 for several ternary water/C14E4/oil systems. The THLB increases with the alkyl chain length of the oil.63 It should be noted that although the values given in Table 3 are theoretical (i.e., neither the presence of surfactant homologues nor the aldehydes in the system are considered), they give an estimate of the variations of THLB values with oil chain length, which was considered appropriate for our purposes. As the oil alkyl chain length increases, the THLB of the system approaches the reaction temperature (i.e., 25 °C). Considering the relationship between the HLB temperature and the interfacial properties (see above), the longer the oil chain length the lower is the oil/water interfacial tension γW/O at 25 °C. This was demonstrated experimentally by measuring the oil/water interfacial tensions at this temperature for the different systems in the presence of phenylacetaldehyde (Table 4). Changes in the oil component may also affect the partition or solvation behavior of the acceptor aldehydes between the continuous (i.e., the oil) and the dispersed (i.e., water) phases.56 The partition coefficients, presented in Table 1, showed that aldehyde 1 was mainly dissolved (62) Kunieda, H.; Shinoda, K. J. Colloid Interface Sci. 1985, 107, 107-121. (63) Shinoda, K.; Kunieda, H. In Encyclopedia of Emulsion Technology; Becher, P., Ed.; Marcel Dekker: New York, 1983; Vol. 1; pp 337367.

Figure 9. Influence of the oil chain length on the equilibrium product yield (a) and initial reaction rate (v°) (b) for the aldolasecatalyzed synthesis of 3 (9) and 4 (b) in W/O gel emulsions of the water/C14E4/oil systems (same composition as in Figure 7) at 25 °C. Aldehydes (50 mM), DHAP (30 mM), and RAMA (0.8 mg/g of emulsion) in 2.5 g of total reaction weight.

in the oil phase while aldehyde 2 partitioned preferentially in the aqueous phase. The product yield at equilibrium and the initial reaction rate (v°) for the model reactions were determined in W/O gel emulsion as a function of the alkyl chain length of the aliphatic hydrocarbon at 25 °C. It should be noted that in all cases the gel emulsions were stable during the reaction. The results, depicted in Figure 9, show that in all cases the longer the oil hydrocarbon chain the better the equilibrium product yields and the faster the initial reaction rate. A thorough inspection of Figure 9 revealed that, in good agreement with the data on the effect of temperature (see previous section), both v° and equilibrium yields were similar for the systems with close THLB regardless of the

Carbon-Carbon Bond Formation in W/O Emulsions

Figure 10. Influence of the partition coefficient on the equilibrium product yield (a) and initial reaction rate (v°) (b) for the aldolase-catalyzed synthesis of 3 (9) and 4 (b) in W/O gel emulsions of the water/C14E4/oil systems (same composition as in Figure 7) at 25 °C. Aldehydes (50 mM), DHAP (30 mM), and RAMA (0.8 mg/g of emulsion) in 2.5 g of total reaction weight.

acceptor aldehyde. This is especially noticeable for the initial reaction rate (v°) (Figure 9b): for instance, water/ C14E4/hexadecane with phenylacetaldehyde (THLB ) 18.1) and water/C14E4/tetradecane with benzyloxyacetaldehyde (THLB ) 17.6) both have similar v° values. The same trend was observed for the systems water/C14E4/tetradecane with phenylacetaldehyde and water/C14E4/dodecane with benzyloxyacetaldehyde and so on. The initial reaction rate v°, akin to the enzymatic activity, had a good correlation with the hydrophobicity

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Figure 11. Influence of water-oil interfacial tension (γ) on the equilibrium product yield (a) and initial reaction rate (v°) (b) for the aldolase-catalyzed synthesis of 3 (9) and 4 (b) in water/C14E4/oil 90/4/6 w/w gel emulsion systems at 25 °C. Aldehydes (50 mM), DHAP (30 mM), and RAMA (0.8 mg/g of emulsion) in 2.5 g of total reaction weight.

(data not shown) of the oil component (i.e., also related to the oil chain length). However, it was thought that log P of the solvent was not the relevant mechanism of the variation of the enzymatic activity with the oil chain length.57 In other words, changes in the enzymatic activity and equilibrium yield due to either the direct interaction between the oil and enzyme or the competition for water molecules can be neglected. The substrate/product solvation and/or partitioning, which are of paramount importance for the reaction equilibrium position and kinetics, and the interfacial properties, are clearly the appropriate parameters.64,65 (64) Reimann, A.; Robb, D. A.; Halling, P. J. Biotechnol. Bioeng. 1994, 43, 1081-1086.

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Plots of the initial reaction rate (v°) and equilibrium product yield versus the partition coefficients and wateroil interfacial tension are depicted in Figures 10 and 11. As can be seen, the lower the water-oil interfacial tension and/or the partition coefficient the higher the initial reaction rate (v°) and equilibrium product yield. It is worth stressing that the values of the water-oil interfacial tension of the W/O gel emulsion systems were measured in the presence of phenylacetaldehyde. However, an identical trend was observed with benzyloxyacetaldehyde. These findings can be rationalized on the basis of the relative changes in the aldehyde substrate solvation in either the continuous phase (i.e., oil phase) or the interfacial regions.57 The partition coefficient is a measure of the relative substrate solvation in the oil and water phases. The lower the partition coefficients the higher the concentration of the aldehyde in the water phase and, therefore, the more favorable the equilibrium position toward product and the higher the initial reaction rates. Moreover, due to the large interfacial area of these systems and the tendency of these substrates to adsorb at the interface, a significant fraction of the aldehydes is affected by the interfacial regions. Thus, the properties of the interface, such as the water/oil interfacial tension, may also change the relative solvation of the aldehydes, the equilibrium yield and reaction kinetics being controlled by this parameter as well. When the interfacial tension of the system is decreased (i.e., when the reaction temperature and THLB are close), the interface becomes less rigid. This also may facilitate the aldehydes to cross the interface affecting the kinetic behavior (i.e., enzymatic activity) and the equilibrium position of the reaction. A question arises whether the partitioning or the interfacial tension is the most important parameter that influences the reaction performance. To assess the dependence level between them, a regression analysis between both the initial reaction rate (v°) and equilibrium yield and both partitioning and interfacial tension was performed.66 The relationship level was measured through the determination coefficient R2 in percent67 that corresponds to the square variations in either v° or equilibrium yield explained by the related variable (i.e., partitioning or interfacial tension). The conclusions were the following. The regression analyses between v° and both variables were highly significant, but the water-oil interfacial (65) Wehtje, E.; Adlercreutz, P.; Mattiasson, B. Biocatal. Biotransform. 1993, 7, 163-176. (66) Statgraphics Plus for Windows, 5th ed.; Manugistics, Inc: Rockville, MD, 2001. (67) Draper, N.; Smith, H. Applied Regression Analysis; John Wiley & Sons: New York, 1981.

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tension gave the highest R2 values: 96-98% of the square variation in v° was explained by this variable. The equilibrium product yield depended on the aldehyde. For the hydrophobic acceptor, phenylacetaldehyde (1), the water-oil interfacial tension explains better than the partitioning (R2 ) 87% versus 78%, respectively), while for the hydrophilic acceptor, benzyloxyacetaldehyde (2), the contrary is true (R2 ) 79% versus 93%, respectively). These results probably reflect that the influence of the interfacial regions on the equilibrium yield is higher for 1 than for 2, which indicates that phenylacetaldehyde is mainly located at the water-oil interface. This fact is also supported by the aforementioned observation concerning the effect of the aldehydes on the HLB temperature of the W/O gel emulsion: the reduction of the THLB was higher for phenylacetaldehyde than that for benzyloxyacetaldehyde, indicating a stronger adsorption of the former on the interface (see Gel Emulsion Formation and Stability). Conclusions Aldolase-catalyzed aldol condensation reactions between DHAP and an acceptor aldehyde were carried out in highly concentrated W/O (gel) emulsions with 90 wt % aqueous component. Neither the donor substrate nor the enzyme produced any change in the properties of the gel emulsions. On the contrary, the aldehyde acceptors decreased the HLB temperature of the system. The initial reaction rate (v°) (i.e., enzymatic activity) and the equilibrium yields depended on the relative changes in the partition of the aldehyde in either the continuous phase (i.e., the oil phase) or interfacial regions. The aldehyde partitioning and the water-oil interfacial tension correlated with both the initial reaction rate and the equilibrium yields. The relative importance of each parameter will depend on the physicochemical properties of the substrates such as their tendency to adsorb at the interface and their hydrophobicity. The equilibrium product yields obtained in conventional DMF/water systems and gel emulsions were similar. Moreover, gel emulsions offer two main advantages. First, the stability of the enzyme in this type of reaction media was much higher (25-fold) than that in aqueous and DMF/buffer media. Second, the amount of solvent used for aldehyde solubilization was considerably reduced from 20% v/v in cosolvent systems to 6 wt % in gel emulsions. Acknowledgment. Financial support from the Spanish C.I.C.Y.T. (Grants QUI99-0997CO2-01 and BIO991219-C02-02) and from Generalitat de Catalunya (Grant 2000SGR-00357) is acknowledged. LA020811B