Transesterification reactions with lipases

Lipases are being used in several reactions of synthesis for the production of valuable compounds. Biodegradable polymers, like butyl oleate and some polyesters, have been synthesized by esterification and transesterification reactions with lipases... 

While diketene remains a very important synthetic precursor, there has been increasing interest in the chemistry of a-methylene-/3-lactones, 3-methylene-2-oxetanones. However, unlike diketene, which can be readily synthesized by the dimerization of aldehydic ketenes, there are few methods for the synthesis of a-methylene-/3-lactones in the literature. Recent strategies for the preparation of the compounds are discussed in Section 2.05.9.2. The kinetic resolution of racemates of alkyl-substituted a-methylene-/3-lactones has been carried out via a lipase-catalyzed transesterification reaction with benzyl alcohol (Equation 21) <1997TA833>. The most efficient lipase tested for this reaction was CAL-B (from Candida antarctica), which selectively transesterifies the (A)-lactone. At 51% conversion, the (R)-f3-lactone, (R)-74, and (A)-/3-hydroxy ester, (S)-75, were formed in very high enantio-selectivities (up to 99% ee). 

In the food industry, lipases are used in lipid modification processes. In these processes the texture, digestibility, or physical properties of natural lipids are modified by lipase-catalyzed transesterification reactions with lipids other than the original fatty acids. In the baking industry, lipases are used to influence the quality of bread through modification of the wheat flour lipids. Finally lipases are used for flavor enhancement of cheese in the dairy industry. 

Other possibilities to prepare chiral cyanohydrins are the enzyme catalysed kinetic resolution of racemic cyanohydrins or cyanohydrin esters [107 and references therein], the stereospecific enzymatic esterification with vinyl acetate [108-111] (Scheme 2) and transesterification reactions with long chain alcohols [107,112]. Many reports describe the use of fipases in this area. Although the action of whole microorganisms in cyanohydrin resolution has been described [110-116],better results can be obtained by the use of isolated enzymes. Lipases from Pseudomonas sp. [107,117-119], Bacillus coagulans [110, 111], Candida cylindracea [112,119,120] as well as lipase AY [120], Lipase PS [120] and the mammalian porcine pancreatic lipase [112, 120] are known to catalyse such resolution reactions. 

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. ... 

Table 2. Catalytic activity of the immobilized lipase in the transesterification reaction of triolein with methanol (18 hours of reaction, 40°C, trioleimmethanol molar ratio 1 3).

In contrast to Mori s synthesis, Pawar and Chattapadhyay used enzymatically controlled enantiomeric separation as the final step [300]. Butanone H was converted into 3-methylpent-l-en-3-ol I. Reaction with trimethyl orthoacetate and subsequent Claisen-orthoester rearrangement yielded ethyl (E)-5-methyl-hept-4-enoate K. Transformation of K into the aldehyde L, followed by reaction with ethylmagnesium bromide furnished racemic ( )-7-methylnon-6-ene-3-ol M. Its enzyme-catalysed enantioselective transesterification using vinylacetate and lipase from Penicillium or Pseudomonas directly afforded 157, while its enantiomer was obtained from the separated alcohol by standard acetylation. 

In a similar investigation, transesterification reactions of vinyl acetate with alcohols in [BMIM]BF4 and [BMIM]PF6 in the presence of immobilized lipases CALB and PS-C were found to proceed with higher enantioselectivities than in THF or toluene, with the best result again being observed with [BMIM]PF6 (280). 

Early reports on the effects of the choice of solvent on enzymatic enantioselectivity showed that substantial changes may be observed. For the transesterification reaction of sec-phenethyl alcohol with vinyl butyrate catalyzed by subtilisin Carlsberg, a 20-fold increase in the E-value was reported when the medium was changed from acetonitrile to dioxane [59]. Similar changes were recorded for the prochiral selectivity of Pseudomonas sp. lipase in the hydrolysis of 2-substituted... 

Lipases, which are noted for their tolerance of organic solvents, were obvious candidates for biocatalysis in ionic liquids. Indeed, stable microbial lipases, such as CaLB [8, 54, 55, 56] and Pseudomonas cepacia lipase (PcL) [28, 55, 57] were cat-alytically active in the ionic liquids of the l-alkyl-3-methylimidazolium and 1-alkylpyridinium families, in combination with anions such as [BF4], [PF6], [TfO] and [ Tf2N]. Early results were not always consistent, which may be caused by impurities that result from the preparation of the ionic liquid. Lipase-mediated transesterification reactions (Figure 10.3) in these ionic liquids proceeded with an efficiency comparable to that in tert-butyl alcohol [8], dioxane [57], or toluene... 

Porcine pancreatic lipase catalyzes the transesterification reaction between tribu-tyrin and various primary and secondary alcohols in a 99% organic medium (Zaks, 1984). Upon further dehydration, the enzyme becomes extremely thermostable. Not only can the dry lipase withstand heating at 100 °C for many hours, but it exhibits a high catalytic activity at that temperature. Reduction in water content also alters the substrate specificity of the lipase in contrast to its wet counterpart, the dry enzyme does not react with bulky tertiary alcohols. 

However, results demonstrated that SCC02 does not show good results for all reactions. It was found for the synthesis of acrylates by transesterification of methylacrylate with lipase from Candida rugosa that owing to its hydrophilic character SCC02 was inferior to more hydrophobic supercritical solvents such as propane, ethane, or even SCSF6 with respect to reactivity (Kamat, 1992). 

We conclude that a commercial immobilized lipase from C. antarctica (Novozym 435) was stable in SCC02 for all experimental conditions investigated. Based on the results obtained here and comparison of them with the results obtained by other investigators, it can be concluded that the magnitude of pressure, temperature, decompression rate, and exposure time needed to inactivate the enzyme strongly depends on the nature and the source of enzyme and, primarily, whether the enzyme is in its native or immobilized form. For the purpose of using this enzyme to catalyze the transesterification reaction of vegetable oils in order to produce esters, the results obtained herein are relevant, because the immobilized lipase can be used with low activity loss at typical conditions of temperature and pressure employed in many biotransformations of raw materials. 

The transesterification reaction can be catalyzed by enzymes, the most common being the lipase. The reaction takes place at normal pressure and temperatures 50 to 55 °C with low energy consumption. The yield of methanolysis depends on several factors as temperature, pH, type of micro-organism producing the enzyme, the use of cosolvents, etc. However, low yields in methyl esters and very long reaction times make the enzymatic processes not competitive enough at this time [9, 11, 17]. 

Microemulsions are used as reaction media for a variety of chemical reactions. The aqueous droplets of water-in-oil micro emulsions can be regarded as minireactors for the preparation of nanoparticles of metals and metal salts and particles of the same size as the starting microemulsion droplets can be obtained [1-3]. Polymerisation in micro emulsions is an efficient way to prepare nanolatexes and also to make polymers of very high molecular weight. Both discontinuous and bicontinuous micro emulsions have been used for the purpose [4]. Microemulsions are also of interest as media for enzymatic reactions. Much work has been done with lipase-catalysed reactions and water-in-oil microemulsions have been found suitable for ester synthesis and hydrolysis, as well as for transesterification [5,6]. 

In the interesterification of fats, 1,3-positional specific lipases catalyze reactions in which only the fatty acids in the a-positions of the triglycerides take part, whereas positional nonspecific lipases are able to catalyze reactions in which the fatty acids from both the a- and / -positions take part. In transesterification between two types of fat, the positional non-specific lipase is therefore able to randomize the fatty acids, resulting in the same fatty acid composition in the triglycerides as obtained in the commercially important chemical randomization process. In ester synthesis, positional non-specific lipases catalyze the reaction with both primary and secondary alcohols whereas positional specific lipases are more or less specific for primary alcohols. 

Acyloxygen fission (63 64), e.g., propiolactone reacts with MeOH, H+ to give 64 (Nu = OMe). A Pseudomonas sp. lipase-promoted asymmetric transesterification reaction allows kinetic resolution of racemic 2-oxetanones <2000J(PI)71>. 

The resulting dynamic nitroaldol system was subsequently challenged with lipase-catalyzed transesterification reactions using different lipases and operational... 

The dynamic cyanohydrin system was next challenged with lipase-catalyzed transesterification resolution using different operational conditions. Thus, different lipases, organic solvents, additives, and acyl donors were evaluated. Isopropenyl acetate 26 was chosen and used as acyl donor because its reaction produces acetone as by-product, which does not interfere in the reaction and the NMR spectra. Molecular sieve 4 A was also added in the dynamic resolution process to control the water activity. The lipase preparation PS-C I was chosen in the resolution process since it expressed the highest lipase activities for both the substrate structure and the enantiomeric selectivities. Different organic solvents were also... 

Table 5.4 Effects of sugars on the enantiomeric ratio (E) and transesterification rate of lipase BC with 6-methyl-5-hepten-2-ol as substrate and toluene as the reaction medium.

Prochiral Compounds. The enantiodifferentiation of prochi-ral compounds by lipase-catalyzed hydrolysis and transesterification reactions is fairly common, with prochiral 1,3-diols most frequently employed as substrates. Recent reports of asymmetric hydrolysis include diesters of 2-substituted 1,3-propanediols and 2-0-protected glycerol derivatives. The asymmetric transesterification of prochiral diols such as 2-0-benzylglycerol and various other 2-substituted 1,3-propanediol derivatives is also fairly common, most frequently with Vinyl Acetate as an irreversible acyl transfer agent. 

The ruthenium-catalyzed racemization of a-methylbenzyl alcohol was combined with an enzyme-catalyzed transesterification with lipase. Dinuclear ruthenium complex 64 effectively catalyzes the racemization of a-methylbenzyl alcohol and the combination of 64, p-chlorophenyl acetate, and enzyme N-435 in the reaction of racemic amethylbenzyl alcohol gave enantiomerically pure (R)-a-methylbenzyl acetate in the excellent yield (Eq. 12.26) [29]. 

Most of lipase-catalyzed acylations of sugars in organic solvents have been reported as transesterification rather than esterification reactions. The displacement of the equilibrium towards products has been accomplished by using activated acyl donors [58] such as 2,2,2-trichloroethyl esters and, more often, enol esters. The use of enol esters, such as a vinyl or an isopropenyl ester, was, in fact, first reported in lipase-catalyzed reactions with sugars [59]. In the reaction, an unstable enol is liberated which instantaneously tautomerizes to the corresponding aldehyde or ketone, making the reaction irreversible. 

Use of Enzymes Lipases are widely used in the processing of fats and oils as catalysts of a number of important lipid reactions, such as hydrolysis, esterification, and transesterification reactions (174). There are a wide number of lipases obtained from different sources, which are available commercially in their free/ crude or immobilized form. However, enzymes with a higher tolerance of pressure would be welcomed, and more research is needed to hopefully develop such enzymes (i.e., genetic engineering or marine sources of the deep ocean). 

Regioselective acylations of polyhydroxylated compounds such as carbohydrates, glycerols, steroids, or alkaloids have been carried out with lipases, esterases, and proteases [13, 20]. One example is the Candida antartica lipase (immobilized on acrylic resin) catalyzed monoacylation of the signalling steroid ectysone (1) giving selectively the 2-C)-acetate 2 (eq. (1)). Using vinyl acetate for this transesterification the reaction was irreversibly pushed to the product side, since the liberated enol instantaneously isomerizes to acetaldehyde [21]. The sometimes unfavorable aldehyde is avoided when 1-ethoxyvinyl acetates [22], trichloro- or -fluoroethyl esters [23 a, b], oxime esters [23 c] or thioesters [23 d] are employed for the quasi-irreversible reaction courses. 

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