Enzymatic transesterification reaction

Figure 9. Enzymatic transesterification reaction of sugar with divinyl...

Kinetic resolution is one of the most important applications of enzymatic transesterification reactions. It is based on the fact that the two enantiomers of a racemic substrate react like two competing reagents. A racemic mixture, often called a racemate, is composed of equal amounts of its two possible enantiomers. The principle of kinetic resolution is shown in Figure 9, where the i -enantiomer [S ] of a racemic substrate is supposed to react faster to the i -product... 

As can be concluded on the basis of Figure 9, only one of the enantiomers reacts in the best case of kinetic resolution. The reaction in the initially racemic mixture [S + S ] then stops at 50% conversion giving 50% of the enantiopure product [P ] and 50% of the enantiopure substrate Enzyme- or chemocat-alyzed racemization of the less reactive enantiomer in situ allows kinetic resolution to be changed to dynamic kinetic resolution when the substrate racemizes fast compared with the product formed and when the enzyme and the product p ) stable under the racemization conditions (Fig. 10). In dynamic kinetic resolution, a racemic mixture is transformed into the product enantiomer with 100% theoretical yield. Since 2003, a kind of revolution has been occurring in the development of dynamic kinetic resolution methods through enzymatic transesterification reactions (15). 

In addition to the catalytic action served by the snRNAs in the formation of mRNA, several other enzymatic functions have been attributed to RNA. Ribozymes are RNA molecules with catalytic activity. These generally involve transesterification reactions, and most are concerned with RNA metabofism (spfic-ing and endoribonuclease). Recently, a ribosomal RNA component was noted to hydrolyze an aminoacyl ester and thus to play a central role in peptide bond function (peptidyl transferases see Chapter 38). These observations, made in organelles from plants, yeast, viruses, and higher eukaryotic cells, show that RNA can act as an enzyme. This has revolutionized thinking about enzyme action and the origin of life itself. 

The synthesis of acrylates from sugars and other substrates has been applied in the early phases of enzymatic reactions and has already been reviewed [2, 14]. The method is attractive because the enzyme allows for mild and, in some examples, stereoselective acrylation. A recent example was published by Popescu et al. who took advantage of the frequent transesterification reactions and reported a route to highly functional linear copoly(meth)acrylates [15]. Methyl (meth)acrylate was mixed with various functional alcohols in the presence of Novozym 435. In situ... 

Contrary to the optical resolutions described in Sections 2.1.1.-2.1.3., which depend on the solubility or chromatographic properties ( Thermodynamic resolution ), the kinetic resolution rests on rate differences shown by the enantiomers when reacted with an optically active reagent. In the ideal case, only one enantiomer is converted into the envisaged product and the other enantiomer is unchanged. In this way, optical resolution is reduced to the more simple separation of two different reaction products. In practice, only two methods of kinetic resolution are reasonably general and reliable the Sharpless epoxidation of allylic alcohols and the enzymatic transesterification of racemic alcohols or carboxylic acids. 

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... 

Based on a suggestion by Odell and Earlam [119] that crown ethers and cryptands can cause proteins to dissolve in methanol, Broos and coworkers [120] investigated the effects of crown ethers on the enzymatic activity of a-chymotrypsin in the transesterification reaction of N-acetyl-L-phenylalanine ethyl ester with n-propanol in organic solvents. They observed a 30-fold rate acceleration when 18-crown-6 was used in octane. At that time, it was proposed that the water- and cation-complexing... 

The enzymatic activity of the L-19 IVS ribozyme results from a cycle of transesterification reactions mechanistically similar to self-splicing. Each ribozyme molecule can process about 100 substrate molecules per hour and is not altered in the reaction therefore the intron acts as a catalyst. It follows Michaelis-Menten kinetics, is specific for RNA oligonucleotide substrates, and can be competitively inhibited. The kcat/Km (specificity constant) is 10s m- 1 s lower than that of many enzymes, but the ribozyme accelerates hydrolysis by a factor of 1010 relative to the uncatalyzed reaction. It makes use of substrate orientation, covalent catalysis, and metalion catalysis—strategies used by protein enzymes. 

Enantioselective enzymatic transesterifications have been used as a complementary method to enantioselective enzymatic ester hydrolyses. The first example of this particular type of biotransformation is the synthesis of the optically active 2-acetoxy-l-silacyclohexane (5 )-78 (Scheme 19). This compound was obtained by an enantioselective transesterification of the racemic l-silacyclohexan-2-ol rac-43 with triacetin (acetate source) in isooctane, catalyzed by a crude lipase preparation from Candida cylindracea (CCL, E.C. 3.1.1.3)62. After terminating the reaction at 52% conversion (relative to total amount of substrate rac-43), the product (S)-78 was separated from the nonreacted substrate by column chromatography on silica gel and isolated in 92% yield (relative to total amount of converted rac-43) with an enantiomeric purity of 95% ee. The remaining l-silacyclohexan-2-ol (/ )-43 was obtained in 76% yield (relative to total amount of nonconverted rac-43) with an enantiomeric purity of 96% ee. Repeated recrystallization of (R)-43 led to an improvement of enantiomeric purity by up to >98% ee. Compound (R)-43 has already earlier been prepared by an enantioselective microbial reduction of the l-silacyclohexan-2-one 42 (see Scheme 8)53. The l-silacyclohexan-2-ol (R)-43 is the antipode of compound (.S j-43 which was obtained by a kinetic enzymatic resolution of the racemic 2-acetoxy-l-silacyclohexane rac-78 (see Scheme 15)62. For further enantioselective enzymatic transesterifications of racemic organosilicon substrates, with a carbon atom as the center of chirality, see References 64 and 70-72. 

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]. 

The reaction is catalyzed by a variety of both acids and bases but simple bases such as NaOH and KOH are generally used for the industrial production of biodiesel [200, 201]. The vegetable oil feedstock, usually soybean or rapeseed oil, needs to be free of water (<0.05%) and fatty acids (<0.5%) in order to avoid catalyst consumption. This presents a possible opportunity for the application of enzymatic transesterification. For example, lipases such as Candida antarctica B lipase have been shown to be effective catalysts for the methanolysis of triglycerides. When the immobilized form, Novozyme 435, was used it could be recycled 50 times without loss of activity [201, 202]. The presence of free fatty acids in the triglyceride did not affect the enzymes performance. The methanolysis of triglycerides catalyzed by Novozyme 435 has also been successfully performed in scC02 as solvent [203]. 

An enantioselectivity of 45% e.e. (at 15% conversion) was observed in the enzymatic transesterification of 2,2,2-trifluoroethyl palmitate by the [60]full-eroproline-derived alcohol ( )-209 (Figure 1.45), catalyzed by lipoprotein lipase (LPL) from Pseudomonas specie.350 The modest enantioselectivity may be related to the distance between the stereogenic center of the substrate and its reactive OH group. If, on the other hand, the group reacting with the enzyme is located closer to the fullerene spheroid, reaction rates slow down... 

As can be seen in Figure 9.4, the enzymatic synthesis of hybrid antioxidants of esculin is feasible in ionic Hquids, resulting in good conversion yields. The conversion yields obtained were higher in ionic liquid [bmim]PF6 as compared to [bmim]BF4 for all phenohc acids tested, while the use of phenohc acid esters in transesterification reactions led to higher conversion yields in both ionic hquid media used. 

Finally, the enzymatic nature of CPIA-cholesterol ester formation will be briefly mentioned. None of the enzyme preparations of three known biosynthetic pathways for cholesterol esters, namely, acyl-CoA cholesterol Q-acyltransferase (ACAT), lecithin cholesterol 0-acyltransferase (LCAT), nor cholesterol esterase, was effective in producing CPIA-cholesterol ester from the Ba isomer or CPIA. In contrast, the 9,000 g supernatant or microsomal fractions from liver or kidney homogenate were found to be capable of producing CPIA-cholesterol ester without the addition of any cofactors. As substrate, only the Ba isomer was effective, and none of the 3 other fenvalerate isomers nor free CPIA was effective. The hepatic enzyme preparation also catalyzed hydrolysis of fenvalerate, and in this case all the 4 isomers were utilized as substrates. These facts imply that CPIA-cholesterol ester is formed from the Ba isomer through a transesterification reaction via intermediary acyl-enzyme complex. 

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. 

Waste oils, from restaurants and household disposals and being creating serious problems of environmental control and food safety, have been considered as good raw material for biodiesel production. Immobilized Candida antarctica lipase was found to be effective for the methanolysis of waste oil. A three-step methanolysis protocol could be used to protect lipase from inactivation by methanol. Compared with one-step reaction, it needs a longer time to reach the reaction equilibrium. So, efforts should be made to increase enzymatic reaction rate. Reports on the enhancement of the activity of certain enzymes by applying ultrasonic irradiation on the enzymes led us to investigate its effects on the enzymatic transesterification of waste oil to biodiesel in a solvent free system. 

A comparative study of biodiesel production with WDO-2 using three-step methanolysis and one-step transesterification with methyl acetate was also conducted here. As can be seen in Figure 5, the ME yield of the three-step methanolysis after reaction for 72 h was 69.1%, which was much lower than those of the one-step transesterification with or without addition of organic base depicted in Figure 4, suggesting that the enzymatic transesterification with methyl acetate was more effective than the enzymatic methanolysis in solvent free system for biodiesel production. 

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