Lipase-catalyzed reactions

The mechanism for the lipase-catalyzed reaction of an acid derivative with a nucleophile (alcohol, amine, or thiol) is known as a serine hydrolase mechanism (Scheme 7.2). The active site of the enzyme is constituted by a catalytic triad (serine, aspartic, and histidine residues). The serine residue accepts the acyl group of the ester, leading to an acyl-enzyme activated intermediate. This acyl-enzyme intermediate reacts with the nucleophile, an amine or ammonia in this case, to yield the final amide product and leading to the free biocatalyst, which can enter again into the catalytic cycle. A histidine residue, activated by an aspartate side chain, is responsible for the proton transference necessary for the catalysis. Another important factor is that the oxyanion hole, formed by different residues, is able to stabilize the negatively charged oxygen present in both the transition state and the tetrahedral intermediate. 

Recently, a very interesting preparation of P-peptides has been carried out by Kanerva and coworkers through a two-step lipase-catalyzed reactions in which N-acetylated P-amino esters were first activated as 2,2,2-trifluoroethyl esters with CALB [55]. The activated esters were then used to react with a P-aminoester in the presence of CALA in dry diethylether or diisopropylether (Scheme 7.31). In this peptide synthesis, the aminoester was used in excess and the unreacted counterpart was easily separated and later recyded. 

The use of ionic liquids (ILs) to replace organic or aqueous solvents in biocatalysis processes has recently gained much attention and great progress has been accomplished in this area lipase-catalyzed reactions in an IL solvent system have now been established and several examples of biotransformation in this novel reaction medium have also been reported. Recent developments in the application of ILs as solvents in enzymatic reactions are reviewed. 

However, the reactions were not enantioselective ones, though the most important aspect of the biocatalysis reaction should be in the enantioselective reaction. We and KragF independently reported the first enantioselective lipase-catalyzed reaction in February-March 2001. Since lipase was anchored by the IL solvent and remained in it after the extraction work-up of the product, we succeeded in demonstrating that recyclable use of the lipase in the [bmim][PFg] solvent system was possible (Fig. 2). ... 

Typically the reaction was carried out as follows to a mixture of lipase in the IL were added this racemic alcohol and vinyl acetate as the acyl donor. The resulting mixture was stirred at 35°C and the reaction course was monitored by GC analysis. After the reaction, ether was added to the reaction mixture to form a biphasic layer, and product acetate and unreacted alcohol were extracted with ether quantitatively. The enzyme remained in the IL phase as expected (Fig. 2). Two months later, Kim and co-workers reported similar results and Lozano and Ibora " reported other examples of lipase-catalyzed reaction in June. Further Park and Kazlauskas reported full details of lipase-catalyzed reaction in an IL solvent... 

Figure 2 Lipase-catalyzed reaction system anchored to the solvent.

We initially tested Candida antarctica lipase using imidazolium salt as solvent because CAL was found to be the best enzyme to resolve our model substrate 5-phenyl-l-penten-3-ol (la) the acylation rate was strongly dependent on the anionic part of the solvents. The best results were recorded when [bmim][BF4] was employed as the solvent, and the reaction rate was nearly equal to that of the reference reaction in diisopropyl ether. The second choice of solvent was [bmim][PFg]. On the contrary, a significant drop in the reaction rate was obtained when the reaction was carried out in TFA salt or OTf salt. From these results, we concluded that BF4 salt and PFg salt were suitable solvents for the present lipase-catalyzed reaction. Acylation of la was accomplished by these four enzymes Candida antarctica lipase, lipase QL from Alcaligenes, Lipase PS from Burkholderia cepacia and Candida rugosa lipase. In contrast, no reaction took place when PPL or PLE was used as catalyst in this solvent system. These results were established in March 2000 but we encountered a serious problem in that the results were significantly dependent on the lot of the ILs that we prepared ourselves. The problem was very serious because sometimes the reaction did not proceed at all. So we attempted to purify the ILs and established a very successful procedure (Fig. 3) the salt was first washed with a mixed solvent of hexane and ethyl acetate (2 1 or 4 1), treated with activated charcoal and passed into activated alumina neutral type I as an acetone solution. It was evaporated and dried under reduced... 

LIPASE-CATALYZED REACTION IN AN IONIC LIQUID SOLVENT SYSTEM... 

One of the most important characteristics of IL is its wide temperature range for the liquid phase with no vapor pressure, so next we tested the lipase-catalyzed reaction under reduced pressure. It is known that usual methyl esters are not suitable for lipase-catalyzed transesterification as acyl donors because reverse reaction with produced methanol takes place. However, we can avoid such difficulty when the reaction is carried out under reduced pressure even if methyl esters are used as the acyl donor, because the produced methanol is removed immediately from the reaction mixture and thus the reaction equilibrium goes through to produce the desired product. To realize this idea, proper choice of the acyl donor ester was very important. The desired reaction was accomplished using methyl phenylth-ioacetate as acyl donor. Various methyl esters can also be used as acyl donor for these reactions methyl nonanoate was also recommended and efficient optical resolution was accomplished. Using our system, we demonstrated the completely recyclable use of lipase. The transesterification took place smoothly under reduced pressure at 10 Torr at 40°C when 0.5 equivalent of methyl phenylthioacetate was used as acyl donor, and we were able to obtain this compound in optically pure form. Five repetitions of this process showed no drop in the reaction rate (Fig. 4). Recently Kato reported nice additional examples of lipase-catalyzed reaction based on the same idea that CAL-B-catalyzed esterification or amidation of carboxylic acid was accomplished under reduced pressure conditions. ... 

Figure 4 Lipase-catalyzed reaction under reduced pressure conditions.

Imidazolium PFg or BF4 salts were frequently used as solvent for the present lipase-catalyzed reaction. However, these salts are very expensive, and we should develop cheaper ILs. Imidazolium alkyl sulfates might be good candidates because various types of alkyl sulfates can be easily prepared. The imidazolium alkyl sulfate was prepared starting from the corresponding ammonium alkyl sulfate as follows ammonium alkyl sulfates ([NH4][RS04]) are easily prepared by the reaction of... 

Lipase-catalyzed reaction is useful for polyester synthesis and IE was employed successfully as solvent. Uyama and Kobayashi demonstrated an efficient polyester synthesis lipase-catalyzed esterification of agipic acid with butan-1,4-diol proceeded smoothly in [bmim][BF4] solvent, particularly under reduced pressure conditions (Fig. 8). Further Russel " and Nara independently reported efficient examples of the lipase-catalyzed polyester synthesis in an IE solvent system. 

Lozano and co-workers reported an interesting stabilization effect of IL for lipase-catalyzed reaction the authors discovered that the presence of an appropriate substrate was essential for stabilization of enzyme in an IL solvent. The half lifetime of native CAL was only 3.2 h in [emim][PFg] solvent, while it lengthened remarkably to 7500 h in the presence of the substrate. The authors succeeded in demonstrating an efficient lipase-recyclable use system based on SCCO2 solvent (Fig. 9). - ... 

Modified enantioselectivity was reported if the reaction was carried out in an IL solvent instead of traditional organic soivent. For instance, Kaziauskas and Kim independently reported that the regioselectivity of lipase-catalyzed reaction was enhanced if the reaction was carried out in an IL solvent system (Fig. 13). " " Recently Wu and co-workers reported another chip of iipase-catalyzed reaction a... 

Figure 11. Lipase-catalyzed reaction in a mixed soivent system using microwave...

Figure 4 Relation between InF and 1/r for the lipase-catalyzed reaction of (1)...

Several mechanisms have been proposed for lipase-catalyzed reactions. Kinetic studies of hydrolysis [14,15] and esterification [50] catalyzed by Pseudomonas cepecia lipase, demonstrate that the enzyme has a ping-pong mechanism. 

The simplest kinetic model applied to describe lipase catalyzed reactions is based on the classic Michaelis-Menten mechanism [10] (Table 3). To test this model Belafi-Bakd et al. [58] studied kinetics of lipase-catalyzed hydrolysis of tri-, di-, and mono-olein separately. All these reactions were found to obey the Michaelis-Menten model. The apparent parameters (K and V ) were determined for global hydrolysis. 

Lipases are able to catalyze many hydrolytic and esterification reactions in the presence of different substrates. The type of substrate is a key factor affecting the activity and productivity of lipase-catalyzed reactions. 

Catalytic site of lipase is known to be a serine-residue and lipase-catalyzed reactions are considered to proceed via an acyl-enzyme intermediate. The mechanism of lipase-catalyzed polymerization of divinyl ester and glycol is proposed as follows (Fig. 3). First, the hydroxy group of the serine residue nucleophilically attacks the acyl-carbon of the divinyl ester monomer to produce an acyl-enzyme intermediate involving elimination of acetaldehyde. The reaction of the intermediate with the glycol produces 1 1 adduct of both... 

In a lipase-catalyzed reaction, the acyl group of the ester is transferred to the hydroxyl group of the serine residue to form the acylated enzyme. The acyl group is then transferred to an external nucleophile with the return of the enzyme to its preacylated state to restart the catalytic cycle. A variety of nucleophiles can participate in this process. For example, reaction in the presence of water results in hydrolysis, reaction in alcohol results in esterification or transesterification, and reaction in amine results in amination. Kirchner et al.3 reported that it was possible to use hydrolytic enzymes under conditions of limited moisture to catalyze the formation of esters, and this is now becoming very popular for the resolution of alcohols.4... 

P. E. Sonnet, M. W. Baillargeon, Methyl-Branched Octanoic Acids as Substrates for Lipase-Catalyzed Reactions , Lipids 1991, 26, 295-300. 

Cull, S. G. Holbrey, J. D. Vargas-Mora, V. et al. Room-temperature ionic liquids as replacements for organic solvents in multiphase bioprocess operations, BiotechnoL Bioeng., 2000, 69(2), 227-233 Lau, R. M. van Rantwijk, F. Seddon, K. R. Sheldon, R. A. Lipase-catalyzed reactions in ionic liquids, Org. Lett., 2000, 2(26), 4189-4191. 

The active site of lipases is characterized by a triad composed of serine, histidine, and aspartate, and acyl-enzyme complexes are the crucial intermediates in all lipase-catalyzed reactions 117,118) (Fig. 13). 

The rate of the reaction in [BDMIM]BF4 was superior to that in [BMIM]BF4. It is significant that no drop in the reaction rate was observed in the [BDMIM]BF4 system after 10 repeated uses of the enzyme, whereas the reaction rate was significantly reduced when the reaction was conducted in [BMIM]BF4 or in [BMIM]PF6 (282). [BDMIM]BF4 was found to be the best solvent for the recycled use of the enzyme under normal pressure conditions when vinyl acetate was used as the acyl donor. No reaction took place when [BDMIM]PF6 was used as the solvent in the lipase-catalyzed reaction. (The [BDMIM]PF6 was purified with particular care to rule out the possibility of contamination.) The replacement of the C2 proton of [BMIM]PF6 by a methyl group was therefore concluded to have a large influence on its biocatalytic compatibility. 

The use of MALDI-MS for the measurement of low molecular mass compounds is widely accepted now [61], but quantification remains problematic. The main problem is the inhomogeneous distribution of the analytes within the matrix [62]. This leads to different amounts of ions and therefore to different signal intensities at various locations of a sample spot. The simplest and most effective way to overcome this problem is the use of an appropriate internal standard [63]. The use of deuterated compounds with a high molecular similarity to the analyte as internal standards leads to a linear correlation between relative signal intensities and relative amount of the compound to be quantified (Fig. 4b) [64]. Using this approach it is possible to quantitate substrates and products of enzyme catalyzed reactions. Two examples were shown recently by Kang and coworkers [64, 65]. The first was a lipase catalyzed reaction which produces 2-methoxy-N-[(lR)-l-phenylethyl]-acetamide (MET) using rac-a-... 

When water molecules interact with an enzyme, it is natural that conformational changes can occur, which in turn can cause changes in the selectivity of the enzyme. Since enantioselectivity of enzymes is of major importance for many applications, it is a common task to investigate how to choose reaction conditions providing the maximal enantioselectivity. As might be expected, because water can interact with enzymes in many ways, it is difficult to generalize the effects. In some studies of lipase-catalyzed esterification reactions, no effects of water activity on enantioselectivity were observed [30]. In a similar study, no effects were observed in most cases, while the enantioselectivity of one lipase-catalyzed reaction decreased... 

C. S. Chen and C. J. Sih, Enantioselective biocatalysis in organic solvents. Lipase catalyzed reactions, Angew. Chem. 1989,... 

Activity maxima at certain aw values have also been found with many other enzymes, such as optima at aw = 0.55 for Mucor miehei lipase-catalyzed reaction in several solvents of different polarity from hexane to 3-pentanone (Valivety, 1992a). In comparing five different lipases, considerable variations not just of the optima of aw but also the dependence of activity on aw itself were found (Valivety, 1992b). Moreover, in polar solvents, correlations of activity with aw often cannot be found at all, probably owing to solvent effects beyond those controlling hydration of the enzyme (Bell, 1997). 

G. Langrand, J. Baratti, G. Buono, and C. Triantaphylides, Lipase catalyzed reactions and strategy for alcohol resolution, Tetrahedron Lett. 1986, 27, 29-32. 

Aaltonen, O. Rantakyla, M. Lipase Catalyzed Reactions of Chiral Compounds in Supercritical Carbon Dioxide In Proceedings of the 2nd International Symposium on Supercritical Fluids, Boston, MA, 1991 pp. 146-149. 

Since lipases are chiral, they possess the ability to distinguish between the two enantiomers of a racemic mixture. The parameter of choice to describe the stereoselectivity or the enantioselectivity of lipase-catalyzed reaction is the enantioselectivity, which is also called the enantiomeric ratio E. The E-value is defined as the ratio of specificity constant for the two enantiomers. 

Liljeblad, A. Lindborg, J. Kanerva, A. Katajisto, J. Kanerva, L. T. Enantioselective lipase-catalyzed reactions of methyl pipecolinate transesterification and N-acylation. Tetrahedron Letters 2002, 43, 2471-2474. 

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