- Email: [email protected]
Effects of crown ethers on the activity of enzymes in peptide formation in organic media#
Stability and Stabilization of Biocatalysts A. Ballesteros, F.J. Plou, J.L. Iborra and P.J. Hailing (Editors) 1998 Elsevier Science B.V.
429
Effects of c r o w n ethers on the activity of e n z y m e s in peptide f o r m a t i o n in organic m e d i a ~ Dirk-Jan van Unen, Johan F.J. Engbersen and David N. Reinhoudt Laboratory of Supramolecular Chemistry and Technology University of Twente, P.O.Box 217, 7500 AE Enschede, The Netherlands Phone +31 53 4892980, Fax +31 53 4894645, E-mail [email protected]
The effects of crown ethers on the rate of protease-catalyzed dipeptide formation using suspensions of oc-chymotrypsin and cross-linked crystals of subtilisin Carlsberg in organic solvents is described. Lyophilisation of cz-chymotrypsin in the presence of 50 equivalents of 18-crown-6 results in a 450 times increased enzymatic activity in acetonitrile. Drying of crosslinked crystalline subtilisin Carlsberg from acetonitrile containing 50 mM 18-crown-6 enhances the activity 13 times. A possible mechanism for the crown ether activation is discussed.
1. INTRODUCTION Nowadays the applicability of enzymes in synthetic organic chemistry is well recognized. Investigations in the field of enzyme-catalyzed organic synthesis were further boosted by the invention that enzymes can function in organic solvents [1]. The use of enzymes in nonaqueous media offer a number of advantages over the use of enzymes in aqueous solutions, like enhanced thermal stability of the enzyme, easy regeneration of the suspended enzyme by filtration, increased substrate solubility, favorable equilibrium shift to synthesis over hydrolysis, and altered selectivity properties of the enzyme [2]. The most important drawback of the use of enzymes in organic media is the reduced activity compared to aqueous conditions. Typically, this reduction in activity is 2 to 6 orders-ofmagnitude. Optimization of the enzymatic activity in organic solvents is therefore one of the major themes of investigation in this field. Modification of charged amino acid residues on the enzymes surface by means of protein engineering or chemical modification is one of the # This investigation was supported by the Netherlands Technology Foundation (STW) and Technical Science Branch of the Netherlands Organization for Advanced and Pure Research (NWO). Dr. I. Sakodinskaya is acknowledged for valuable discussions.
430 strategies. Furthermore, immobilization and medium engineering were shown to result in increased enzyme performance. We have reported that addition of crown ethers enhances the activity of proteases [3] and tyrosinases [4] in organic solvents. High accelerations, typically in the range of 500 times, were found when the crown ether was added prior to lyophilisation [5]. Thiacrown ethers were also reported to change the enantioselectivity of lipases [6]. The low enzymatic activity in organic solvents is one of the major reasons why applications on a larger scale are still a future prospective. A reaction which is potentially very suitable for application of enzymes in organic solvents is the enzymatic synthesis of peptide bonds. The formation of peptide bonds catalyzed by proteases offers clear advantages over chemical methods, such as mild reaction conditions, no racemization, and the fact that time-consuming protection and deprotection steps render superfluous. It is obvious that in order to overcome hydrolysis problems this reaction needs to be performed in non-aqueous systems. Here, we report a study of the protease-catalyzed formation of N-acetyl-L-phenylalanyl-Lphenylalaninamide from the 2-chloroethyl ester of N-acetyl-L-phenylalanine and Lphenylalaninamide and the effects of crown ethers on this synthesis. The effects are studied using suspensions of tx-chymotrypsin and subtilisin Carlsberg enzyme crystals.
2. MATERIALS AND METHODS tx-Chymotrypsin (E.C. 3.4.21.1) type 11 from Bovine Pancreas (54 U/mg protein; hydrolysis of N-benzoyl-L-tyrosine ethyl ester at pH 7.8) and the peptide precursors N-Ac-L-Phe and LPhe-NH2 were obtained from Sigma (St. Louis, MO). Cross-linked crystals of subtilisin Carlsberg were prepared according to the method of Schmitke et al. [7]. The 2chloroethylester of N-Ac-L-Phe was synthesized from N-Ac-L-Phe and 2-chloroethanol using Amberlite IR-120 as a catalyst. 18-Crown-6 was a gift from Shell Laboratories (Amsterdam, The Netherlands). The solvents were of analytical grade or higher and were from Acros (Geel, Belgium).
2.1. Pretreatment of o~.chymotrypsin a-Chymotrypsin (5 mg/ml) was dissolved in 20 mM sodium phosphate buffer pH 7.8 containing the appropriate amount of 18-crown-6. The equivalents of added crown ether are relative to the moles of enzyme. The samples were lyophilized, after rapid freezing in liquid nitrogen, for 24 hours. For comparison studies D-sorbitol (5 mg/ml) was used instead of crown ether, using the similar procedure. 2.2. Pretreatment of cross-linked crystalline subtilisin Carisberg Cross-linked crystals of subtilisin Carlsberg (1.0 mg/ml) were soaked in acetonitrile containing the indicated concentration of 18-crown-6. Subsequently the solvent was left to evaporate overnight at room temperature.
431 2.3. Studies on the effect of 18-crown-6 on the rate of dipeptide formation All enzyme preparations, peptide precursors and solvents were equilibrated at a thermodynamic water activity of 0.113 above a saturated LiC1 solution for 24 hours. Reactions were performed in duplicate on a 1 ml scale with magnetic stirring at 500 rev./min. Typical conditions: 2.5 mg/ml r or 0.5 mg/ml subtilisin Carlsberg enzyme crystals, 50 mM N-Ac-L-Phe-OEtC1 and 50 mM L-Phe-NH2 in acetonitrile at 30~ The reactions were terminated by the addition of 4 volumes of dimethylsulphoxide. The reaction mixture was analyzed by HPLC. Initial rates were calculated from conversions <5%.
3. RESULTS AND DISCUSSION The reaction studied was the protease-catalyzed peptide bond formation between the 2chloroethylester of N-acetyl-L-phenylalanine and L-phenylalaninamide, using (x-chymotrypsin and cross-linked crystalline subtilisin Carlsberg (Scheme 1).
~
~
+
O
'~N
C! O
N-Ac-L-Phe-OEtC1
~
protease
0
,,Jr,.
0
H2N
O
L-Phe-NH 2
N-Ac-L-Phe-L-Phe-NH 2
Scheme 1. Protease-catalyzed peptide bond formation. 3.1. Effects of 18-crown-6 on peptide bond formation using o~-chymotrypsin Initially, the catalytic activity of three proteases, namely subtilisin Carlsberg, trypsin and r chymotrypsin, present as suspensions in acetonitrile was investigated for the reaction depicted in Scheme 1. All three proteases could be activated by lyophilisation in the presence of 18crown-6. The highest acceleration in the rate of peptide bond formation was found in the case of r Therefore, further studies were performed using this enzyme. In order to optimize the activation effect, the influence of the amount of 18-crown-6 was studied (Table 1). In the presence of 50 to 100 equivalents of crown ether a maximum activity enhancement of 450 times is observed. This results in an enzymatic activity of 0.7 U/mg enzyme. Upon addition of larger amounts of crown ether the enzyme activation is decreasing. The activation effect is due to the macrocyclic nature of the crown ether while pentaglyme, the linear chain analog of 18-crown-6, has no effect on the enzyme activity. Comparative studies using D-sorbitol, a well-known lyoprotectant, instead of crown ether revealed that the
432 activation effect of 18-crown-6 was much more effective as pretreatment with D-sorbitol resulted in an acceleration of only 8 times. Table 1 Crown ether induced enhancement (V0(18-crown-6)/V0) of the ct-chymotrypsin-catalyzed peptide bond formation between N-Ac-L-Phe-OEtC1 and L-Phe-NH2 in acetonitrile. For details, see methods. Equivalents of 18-crown-6
V0
Acceleration
0 50 100 250 500 1000 2500
(nmol/min*mg protein ) 1.5 674 700 462 130 12.4 0.8
436 450 298 84 8 0.5
A hypothesis for the mechanism of the crown ether acceleration effect is that 18-crown-6 is complexing the e-ammonium functions of the lysine residues on the exterior of the enzyme. In this way protonation of the amine functions is stabilized. Also the formation of inter- and intramolecular salt-bridges between anionic sites on the enzyme and these ammonium functions in organic solvents is prevented. Formation of these salt-bridges may result in enzyme molecules with altered catalytic properties compared to the native enzyme. The most direct proof that the ammonium functions play a role in the crown ether acceleration is the observation that acetylated trypsin, in which the lysine residues have been acetylated, are not activated by crown ether pretreatment, while normal trypsin is accelerated very effectively [5]. When larger amounts of crown ethers are added one can imagine that also polar residues from the interior of the enzyme are complexed, resulting in a deformation of the native enzyme conformation and a lower catalytic activity. Conformational changes of the enzyme during lyophilisation are known to occur. Upon suspending the lyophilized enzyme powder into an organic solvent the enzyme can be locked in a distorted conformation due to lack of conformational freedom. This may explain the observed decrease in enzyme activaty at larger amounts of 18-crown-6. The crown ether induced enhancement of enzyme activity also turned out to be applicable for peptide bond formation using a variety of peptide precursors, like N-acetyl-L-tyrosine ethylester, D-phenylalaninamide, L-leucinamide, and L-tyrosinamide, and in a whole range of solvents, ranging in polarity from dioxane to toluene.
433 3.2. Effects of 18-crown-6 on peptide bond formation using cross-linked enzyme crystals of subtilisin Carlsberg An emerging technology in the field of non-aqueous enzymology is the use of cross-linked crystalline enzymes [8,9]. These mechanically and thermally stable class of biocatalysts were investigated on the effects of crown ethers. Addition of 18-crown-6 to the reaction mixture without pretreatment of the subtilisin Carlsberg crystals did not result in any acceleration. The initial peptide formation activity remained 0.44 nmol/min*mg enzyme. As freeze drying of enzyme crystals from aqueous buffer results in a distortion of the enzyme crystal lattice, due to crystallisation of the water during freezing, lyophilisation in the presence of crown ethers, as was performed for the r preparations in the previous section, is not expected to be a suitable procedure in this case. Therefore, another way of pretreatment with crown ethers has been investigated, that is soaking and drying of cross-linked crystalline subtilisin Carlsberg from an organic solvent, typically acetonitrile, in which 18-crown-6 is dissolved.
14 "N
12 10
<
/
8
0
J
i0
/
Y
z0
3o
.io
5o
[ 18-crown-6], (raM) Figure 1. Effect of the amount of 18-crown-6 on the peptide bond formation between N-AcL-Phe-OEtC1 and L-Phe-NH2 in acetonitrile using soaked cross-linked crystalline subtilisin Carlsberg. For details, see methods. Figure 1 showes that soaking and drying of the enzyme crystals results in a enhancement of the enzymatic activity. An acceleration of 13 times can be achieved by drying the enzyme crystals in the presence of 50 mM 18-crown-6. Drying of cross-linked crystalline enzymes in the presence of surfactants was also reported [9] to enhance enzymatic activity. However, most surfactants lost their activating effect after storage for a few days. In contrast, the crown ether activation turned out to be very stable as the activated cross-linked crystals had still the same activity after one month of storage at 4~ In conclusion, these results show that lyophilisation of t~-chymotrypsin and soaking of crosslinked crystalline subtilisin Carlsberg with 18-crown-6 results in enzyme preparations with
434 strongly enhanced catalytic properties in the peptide bond formation in organic solvents. Since crown ethers are readily available and the pretreatment procedure is easily performed this approach is a versatile way to overcome the usually observed large decrease in activity of enzymes in organic media.
REFERENCES
1. J.S. Dordick, Enzyme Microb. Technol., 11 (1989) 194. 2. A.M.P. Koskinen and A.M. Klibanov (eds.), Enzymatic reactions in organic media, Blackie academic & professional, Glasgow, 1996. 3. D.N. Reinhoudt, A.M. Eendebak, W.F. Nijenhuis, W. Verboom, M. Kloosterman and H.E. Schoemaker, J. Chem. Soc., Chem. Commun., (1989) 399; J. Broos, M.N. Martin, I. Rouwenhorst, W. Verboom and D.N. Reinhoudt, Recl. Trav. Chim. Pays-Bas, 110 (1991) 222; J.F.J. Engbersen, J. Broos, W. Verboom and D.N. Reinhoudt, Pure & Appl. Chem., 68 (1996) 2171. 4. J. Broos, R. Arends, G.B. van Dijk, W. Verboom., J.F.J. Engbersen and D.N. Reinhoudt, J. Chem. Soc., Perkin Trans. 1, (1996) 1415. 5. J. Broos, I.K. Sakodinskaya, J.F.J. Engbersen, W. Verboom and D.N. Reinhoudt, J. Chem. Soc., Chem. Commun., (1995) 255. 6. T. Itoh, Y. Takagi, T. Murakami, Y. Hiyama and H. Tsukube, J. Org. Chem., 61 (1996) 2158; Y. Takagi, J. Teramoto, H. Kihara, T. Itoh and H. Tsukube, Tetrahedron Lett., 37 (1996) 4991. 7. J.L. Schmitke, C.R. Wescott and A.M. Klibanov, J. Am. Chem. Soc., 118 (1996) 3360. 8. N.L. St. Clair and M.A. Navia, J. Am. Chem. Soc., 114 (1992) 7314. 9. N. Khalaf, C.P. Govardhan, J.J. Lalonde, R.A. Persichetti, Y.-F. Wang and A.L. Margolin, J. Am. Chem. Soc., 118 (1996) 5494.
429
Effects of c r o w n ethers on the activity of e n z y m e s in peptide f o r m a t i o n in organic m e d i a ~ Dirk-Jan van Unen, Johan F.J. Engbersen and David N. Reinhoudt Laboratory of Supramolecular Chemistry and Technology University of Twente, P.O.Box 217, 7500 AE Enschede, The Netherlands Phone +31 53 4892980, Fax +31 53 4894645, E-mail [email protected]
The effects of crown ethers on the rate of protease-catalyzed dipeptide formation using suspensions of oc-chymotrypsin and cross-linked crystals of subtilisin Carlsberg in organic solvents is described. Lyophilisation of cz-chymotrypsin in the presence of 50 equivalents of 18-crown-6 results in a 450 times increased enzymatic activity in acetonitrile. Drying of crosslinked crystalline subtilisin Carlsberg from acetonitrile containing 50 mM 18-crown-6 enhances the activity 13 times. A possible mechanism for the crown ether activation is discussed.
1. INTRODUCTION Nowadays the applicability of enzymes in synthetic organic chemistry is well recognized. Investigations in the field of enzyme-catalyzed organic synthesis were further boosted by the invention that enzymes can function in organic solvents [1]. The use of enzymes in nonaqueous media offer a number of advantages over the use of enzymes in aqueous solutions, like enhanced thermal stability of the enzyme, easy regeneration of the suspended enzyme by filtration, increased substrate solubility, favorable equilibrium shift to synthesis over hydrolysis, and altered selectivity properties of the enzyme [2]. The most important drawback of the use of enzymes in organic media is the reduced activity compared to aqueous conditions. Typically, this reduction in activity is 2 to 6 orders-ofmagnitude. Optimization of the enzymatic activity in organic solvents is therefore one of the major themes of investigation in this field. Modification of charged amino acid residues on the enzymes surface by means of protein engineering or chemical modification is one of the # This investigation was supported by the Netherlands Technology Foundation (STW) and Technical Science Branch of the Netherlands Organization for Advanced and Pure Research (NWO). Dr. I. Sakodinskaya is acknowledged for valuable discussions.
430 strategies. Furthermore, immobilization and medium engineering were shown to result in increased enzyme performance. We have reported that addition of crown ethers enhances the activity of proteases [3] and tyrosinases [4] in organic solvents. High accelerations, typically in the range of 500 times, were found when the crown ether was added prior to lyophilisation [5]. Thiacrown ethers were also reported to change the enantioselectivity of lipases [6]. The low enzymatic activity in organic solvents is one of the major reasons why applications on a larger scale are still a future prospective. A reaction which is potentially very suitable for application of enzymes in organic solvents is the enzymatic synthesis of peptide bonds. The formation of peptide bonds catalyzed by proteases offers clear advantages over chemical methods, such as mild reaction conditions, no racemization, and the fact that time-consuming protection and deprotection steps render superfluous. It is obvious that in order to overcome hydrolysis problems this reaction needs to be performed in non-aqueous systems. Here, we report a study of the protease-catalyzed formation of N-acetyl-L-phenylalanyl-Lphenylalaninamide from the 2-chloroethyl ester of N-acetyl-L-phenylalanine and Lphenylalaninamide and the effects of crown ethers on this synthesis. The effects are studied using suspensions of tx-chymotrypsin and subtilisin Carlsberg enzyme crystals.
2. MATERIALS AND METHODS tx-Chymotrypsin (E.C. 3.4.21.1) type 11 from Bovine Pancreas (54 U/mg protein; hydrolysis of N-benzoyl-L-tyrosine ethyl ester at pH 7.8) and the peptide precursors N-Ac-L-Phe and LPhe-NH2 were obtained from Sigma (St. Louis, MO). Cross-linked crystals of subtilisin Carlsberg were prepared according to the method of Schmitke et al. [7]. The 2chloroethylester of N-Ac-L-Phe was synthesized from N-Ac-L-Phe and 2-chloroethanol using Amberlite IR-120 as a catalyst. 18-Crown-6 was a gift from Shell Laboratories (Amsterdam, The Netherlands). The solvents were of analytical grade or higher and were from Acros (Geel, Belgium).
2.1. Pretreatment of o~.chymotrypsin a-Chymotrypsin (5 mg/ml) was dissolved in 20 mM sodium phosphate buffer pH 7.8 containing the appropriate amount of 18-crown-6. The equivalents of added crown ether are relative to the moles of enzyme. The samples were lyophilized, after rapid freezing in liquid nitrogen, for 24 hours. For comparison studies D-sorbitol (5 mg/ml) was used instead of crown ether, using the similar procedure. 2.2. Pretreatment of cross-linked crystalline subtilisin Carisberg Cross-linked crystals of subtilisin Carlsberg (1.0 mg/ml) were soaked in acetonitrile containing the indicated concentration of 18-crown-6. Subsequently the solvent was left to evaporate overnight at room temperature.
431 2.3. Studies on the effect of 18-crown-6 on the rate of dipeptide formation All enzyme preparations, peptide precursors and solvents were equilibrated at a thermodynamic water activity of 0.113 above a saturated LiC1 solution for 24 hours. Reactions were performed in duplicate on a 1 ml scale with magnetic stirring at 500 rev./min. Typical conditions: 2.5 mg/ml r or 0.5 mg/ml subtilisin Carlsberg enzyme crystals, 50 mM N-Ac-L-Phe-OEtC1 and 50 mM L-Phe-NH2 in acetonitrile at 30~ The reactions were terminated by the addition of 4 volumes of dimethylsulphoxide. The reaction mixture was analyzed by HPLC. Initial rates were calculated from conversions <5%.
3. RESULTS AND DISCUSSION The reaction studied was the protease-catalyzed peptide bond formation between the 2chloroethylester of N-acetyl-L-phenylalanine and L-phenylalaninamide, using (x-chymotrypsin and cross-linked crystalline subtilisin Carlsberg (Scheme 1).
~
~
+
O
'~N
C! O
N-Ac-L-Phe-OEtC1
~
protease
0
,,Jr,.
0
H2N
O
L-Phe-NH 2
N-Ac-L-Phe-L-Phe-NH 2
Scheme 1. Protease-catalyzed peptide bond formation. 3.1. Effects of 18-crown-6 on peptide bond formation using o~-chymotrypsin Initially, the catalytic activity of three proteases, namely subtilisin Carlsberg, trypsin and r chymotrypsin, present as suspensions in acetonitrile was investigated for the reaction depicted in Scheme 1. All three proteases could be activated by lyophilisation in the presence of 18crown-6. The highest acceleration in the rate of peptide bond formation was found in the case of r Therefore, further studies were performed using this enzyme. In order to optimize the activation effect, the influence of the amount of 18-crown-6 was studied (Table 1). In the presence of 50 to 100 equivalents of crown ether a maximum activity enhancement of 450 times is observed. This results in an enzymatic activity of 0.7 U/mg enzyme. Upon addition of larger amounts of crown ether the enzyme activation is decreasing. The activation effect is due to the macrocyclic nature of the crown ether while pentaglyme, the linear chain analog of 18-crown-6, has no effect on the enzyme activity. Comparative studies using D-sorbitol, a well-known lyoprotectant, instead of crown ether revealed that the
432 activation effect of 18-crown-6 was much more effective as pretreatment with D-sorbitol resulted in an acceleration of only 8 times. Table 1 Crown ether induced enhancement (V0(18-crown-6)/V0) of the ct-chymotrypsin-catalyzed peptide bond formation between N-Ac-L-Phe-OEtC1 and L-Phe-NH2 in acetonitrile. For details, see methods. Equivalents of 18-crown-6
V0
Acceleration
0 50 100 250 500 1000 2500
(nmol/min*mg protein ) 1.5 674 700 462 130 12.4 0.8
436 450 298 84 8 0.5
A hypothesis for the mechanism of the crown ether acceleration effect is that 18-crown-6 is complexing the e-ammonium functions of the lysine residues on the exterior of the enzyme. In this way protonation of the amine functions is stabilized. Also the formation of inter- and intramolecular salt-bridges between anionic sites on the enzyme and these ammonium functions in organic solvents is prevented. Formation of these salt-bridges may result in enzyme molecules with altered catalytic properties compared to the native enzyme. The most direct proof that the ammonium functions play a role in the crown ether acceleration is the observation that acetylated trypsin, in which the lysine residues have been acetylated, are not activated by crown ether pretreatment, while normal trypsin is accelerated very effectively [5]. When larger amounts of crown ethers are added one can imagine that also polar residues from the interior of the enzyme are complexed, resulting in a deformation of the native enzyme conformation and a lower catalytic activity. Conformational changes of the enzyme during lyophilisation are known to occur. Upon suspending the lyophilized enzyme powder into an organic solvent the enzyme can be locked in a distorted conformation due to lack of conformational freedom. This may explain the observed decrease in enzyme activaty at larger amounts of 18-crown-6. The crown ether induced enhancement of enzyme activity also turned out to be applicable for peptide bond formation using a variety of peptide precursors, like N-acetyl-L-tyrosine ethylester, D-phenylalaninamide, L-leucinamide, and L-tyrosinamide, and in a whole range of solvents, ranging in polarity from dioxane to toluene.
433 3.2. Effects of 18-crown-6 on peptide bond formation using cross-linked enzyme crystals of subtilisin Carlsberg An emerging technology in the field of non-aqueous enzymology is the use of cross-linked crystalline enzymes [8,9]. These mechanically and thermally stable class of biocatalysts were investigated on the effects of crown ethers. Addition of 18-crown-6 to the reaction mixture without pretreatment of the subtilisin Carlsberg crystals did not result in any acceleration. The initial peptide formation activity remained 0.44 nmol/min*mg enzyme. As freeze drying of enzyme crystals from aqueous buffer results in a distortion of the enzyme crystal lattice, due to crystallisation of the water during freezing, lyophilisation in the presence of crown ethers, as was performed for the r preparations in the previous section, is not expected to be a suitable procedure in this case. Therefore, another way of pretreatment with crown ethers has been investigated, that is soaking and drying of cross-linked crystalline subtilisin Carlsberg from an organic solvent, typically acetonitrile, in which 18-crown-6 is dissolved.
14 "N
12 10
<
/
8
0
J
i0
/
Y
z0
3o
.io
5o
[ 18-crown-6], (raM) Figure 1. Effect of the amount of 18-crown-6 on the peptide bond formation between N-AcL-Phe-OEtC1 and L-Phe-NH2 in acetonitrile using soaked cross-linked crystalline subtilisin Carlsberg. For details, see methods. Figure 1 showes that soaking and drying of the enzyme crystals results in a enhancement of the enzymatic activity. An acceleration of 13 times can be achieved by drying the enzyme crystals in the presence of 50 mM 18-crown-6. Drying of cross-linked crystalline enzymes in the presence of surfactants was also reported [9] to enhance enzymatic activity. However, most surfactants lost their activating effect after storage for a few days. In contrast, the crown ether activation turned out to be very stable as the activated cross-linked crystals had still the same activity after one month of storage at 4~ In conclusion, these results show that lyophilisation of t~-chymotrypsin and soaking of crosslinked crystalline subtilisin Carlsberg with 18-crown-6 results in enzyme preparations with
434 strongly enhanced catalytic properties in the peptide bond formation in organic solvents. Since crown ethers are readily available and the pretreatment procedure is easily performed this approach is a versatile way to overcome the usually observed large decrease in activity of enzymes in organic media.
REFERENCES
1. J.S. Dordick, Enzyme Microb. Technol., 11 (1989) 194. 2. A.M.P. Koskinen and A.M. Klibanov (eds.), Enzymatic reactions in organic media, Blackie academic & professional, Glasgow, 1996. 3. D.N. Reinhoudt, A.M. Eendebak, W.F. Nijenhuis, W. Verboom, M. Kloosterman and H.E. Schoemaker, J. Chem. Soc., Chem. Commun., (1989) 399; J. Broos, M.N. Martin, I. Rouwenhorst, W. Verboom and D.N. Reinhoudt, Recl. Trav. Chim. Pays-Bas, 110 (1991) 222; J.F.J. Engbersen, J. Broos, W. Verboom and D.N. Reinhoudt, Pure & Appl. Chem., 68 (1996) 2171. 4. J. Broos, R. Arends, G.B. van Dijk, W. Verboom., J.F.J. Engbersen and D.N. Reinhoudt, J. Chem. Soc., Perkin Trans. 1, (1996) 1415. 5. J. Broos, I.K. Sakodinskaya, J.F.J. Engbersen, W. Verboom and D.N. Reinhoudt, J. Chem. Soc., Chem. Commun., (1995) 255. 6. T. Itoh, Y. Takagi, T. Murakami, Y. Hiyama and H. Tsukube, J. Org. Chem., 61 (1996) 2158; Y. Takagi, J. Teramoto, H. Kihara, T. Itoh and H. Tsukube, Tetrahedron Lett., 37 (1996) 4991. 7. J.L. Schmitke, C.R. Wescott and A.M. Klibanov, J. Am. Chem. Soc., 118 (1996) 3360. 8. N.L. St. Clair and M.A. Navia, J. Am. Chem. Soc., 114 (1992) 7314. 9. N. Khalaf, C.P. Govardhan, J.J. Lalonde, R.A. Persichetti, Y.-F. Wang and A.L. Margolin, J. Am. Chem. Soc., 118 (1996) 5494.