Lipase catalyzed esterification

Orthoformates have been used in the lipase-catalyzed esterification aimed at the kinetic resolution of racemic acids such as flurbiprofen, a nonsteroidal anti-inflammatory drug (Figure 6.18). Orthoformates trap the water as it is formed through hydrolysis, and therefore prevent the reverse reaction, and, at the same time, provide the alcohol for the esteriflcation [65]. 

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. 

The lipases demonstrated very high stability in media partially or totally composed of organic solvent. In such media, the lipases catalyze esterification, transesterification, and resolution of enantiomers [19,45,75,97-100]. Nevertheless, several biphasic systems (organic-aqueous) are used for hydrolysis of lipid and fats [7,34,101]. Kinetic studies in biphase media or in inverted micelles demonstrate that the lipase behavior is different... 

Yan, Y.C., Bornscheuer, U.T. and Schmid, R.D. (2002) Efficient water removal in lipase-catalyzed esterifications using a low-boiling-point azeotrope. Biotechnology and Bioengineering, 78 (1), 31-34. 

In lipase-catalyzed esterifications and transesterifications, esters of halo-genated alcohols, typically 2-chloroethanol, 2,2,2-trifluoroethanol, and 2,2,2-... 

Promotion of Lipase-Catalyzed Esterification of N-Valeric Acid and Citro-neUol in Supercritical Carbon Dioxide in the Near-Critical Region (Ikushima et al 1996). 

Ester synthesis of fatty acid ethyl ester. The lipase-catalyzed esterification of fatty acid and alcohol is well-known. It was also favorable for the esterification of poly unsaturated fatty acids under mild conditions with the enzyme. However, the activity of native lipase is lower in polar organic solvents, i.e. ethanol and methanol. The synthesis of Ae fatty acid ethyl ester was carried out in ethanol using the palmitic acid-modified lipase. As shown in Figure 7, the reactivity of the modified lipase in this system was much higher than that of the unmoditied lipase. 

Relatively few detailed studies of enzyme kinetics in organic media have been carried out. Preferably, full kinetics should be studied, allowing the determination of Km and kcat values, but it is much more common to see just reports on the catalytic activity at fixed substrate concentrations as a function of water activity. That such studies can be misleading was shown in an investigation of lipase-catalyzed esterification [26]. When the reaction rate in the esterification reaction was plotted versus the water activity at three different substrate concentrations, maxima were obtained at three different water activities (Figure 1.4). Such maxima should not be used to claim that the optimal water activity of the enzyme was found. Detailed kinetic studies showed that both the kcat and the Km values (for the alcohol substrate) varied with the water activity. The Km value of the alcohol increased with increasing water... 

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... 

When substrate activities are used instead of substrate concentrations in studies of enzyme kinetics in organic media, solvent effects due to substrate solvation disappear. Remaining solvent effects should be due to direct interactions between the enzyme and the solvent. In a study of lipase-catalyzed esterification reactions, it was found that Km values based on activities were indeed more similar tban those based on concentrations in different solvents, but still some differences remained [49]. 

It is clear that the water activity is of crucial importance for the equilibrium yield in a reversed hydrolysis reaction. As expected, the equilibrium yield increases with decreasing water activity. This has been shown, for example, for the condensation of glucose and octanol [62], esterification of lysophospholipids with fatty acids [29, 63], and in normal lipase-catalyzed esterification reactions [64, 65]. The same situation is observed in ionic liquids [66]. 

Following the first examples of the acylation of adenosine and uridine [2], the regioselective lipase-catalyzed esterification of these important molecules has been extensively investigated by Gotor s group [21g]. Attention has been mainly focused on deoxynucleosides - compounds of special interest in medicinal chemistry because of their antiviral and antitumoral properties. Some significant examples are discussed below. 

Another emerging technique which deserves mention is the use of a supported ionic liquid membrane. This involves two liquid phases that both contain an enzyme and are separated by the membrane. Lipase-catalyzed esterification takes place in the feed phase to afford a mixture of the (R)-acid and the (S)-ester (Figure 10.22). The latter diffuses through the membrane and is hydrolyzed in the receiving phase to afford the (S)-acid [151, 152]. The methodology has been applied, for example, to the resolution of ibuprofen [151]. 

It is known that enantioselectivity of enzymes depends on many different parameters such as temperature, substrate structure, reaction medium, and presence of water. Enantiopreference of enzymes can be greatly affected, even reversed, by changing the reaction solvent. Such an example was reported by Ueji et al. in 1992 for Candida cylindracea lipase-catalyzed esterification of ( )-2-phenoxy propionic acid with 1-butanol [29]. 

G. Durand, Comparison of lipase-catalyzed esterification in supercritical carbon dioxide and in u-hexane, Biotechnol. Lett. 1990, 12, 11-16. 

Knez, Z. Habulin, M. Lipase Catalyzed Esterification in Supercritical Carbon Dioxide. In Biocatalysis in Non-Conventional Media Tramper, J., Vermiie, M. H., Beeftink, H. H., Eds. Elsevier Science Amsterdam, 1992, pp. 401-406. Knez, Z. Habulin, M. Krmelj, V. Enzyme Catalyzed Reactions in Dense Gases. J. Supercrit. Fluids 1998, 14, 17-29. 

Marty, A. Chulalaksananukul, W. Willemot, R. M. Condoret, J. S. Kinetics of Lipase-Catalyzed Esterification in Supercritical C02. Biotechnol. Bioeng. 1992b, 39, 273-280. 

Figure 31 Lipase-catalyzed esterification of 2-(2-fluoro-4-biphenyl)propanoic acid 56 with various alcohols.

The resolution of (7i,. S )-naproxen 54 using lipase-catalyzed esterification of the free acid with different diols and organic solvents was reported. The (.S)-naproxcn ester 55 was obtained in > 99 % ee when using 1,4-butandiol.79... 

The objective of the present work was to study the synthesis of monolaurin by direct lipase-catalyzed esterification between glycerol and lauric acid without any solvent or surfactant. The effects of lauric acid/ glycerol molar ratio, enzyme concentration, and temperature were studied using an experimental design. The reuse of the commercial immobilized lipase, to reduce the process cost, was also investigated. 

Reetz MT, Wiesenhofer W (2004) Liquid polyethylene glycol) and supercritical carbon dioxide as a biphasic solvent system for lipase-catalyzed esterification. Chem Commun 23 2750-2751... 

Lipase catalyzed esterifications were performed favorably solvent-free [21] and were applied also to bulk polymerization for polyester synthesis [22]. 

Purified PUFAs can be converted to their ethyl esters by adopting lipase-catalyzed esterification. For example, the ethyl esterification of DHA (Shimada et al., 2001b, 2003d) with an equal mol of EtOH using immobilized C. antarc-... 

Table 1. Enantiomer Ratio and Yield (in brackets) of Lipase-Catalyzed Esterification of 70a,b...

Fig. 5 1 H-NMR spectra of (a) CDS-2 at equilibrium in the absence of lipase and acyl donor and (b) lipase-catalyzed esterification resolution of dynamic system after 14 days. Compound numbering as in Scheme 9. Modified from [15], copyright Wiley-VCH Verlag GmbH Co. KGaA. Reproduced with permission...

Figure 7.2 The structure of the faster reacting enantiomer in lipase-catalyzed esterification in kinetic resolution of racemic secondary alcohols or hydrolysis of the corresponding esters. Small and large refer to the relative size of the groups and not to the R/S notation.

As an example of the use of SC-CO2 in an enzymatic reaction, the lipase-catalyzed esterification of oleic acid with racemic ( )-citronellol should be mentioned. At 31 °C and 8.4 MPa, the (—)-(5)-ester is formed enantioselectively in SC-CO2 with an optical purity of nearly 100% [924]. The reaction rate is enhanced by increasing pressure, i.e. by increasing the solvation capability or solvent polarity of SC-CO2. A linear correlation has been found between reaction rate and the solvatoehromie solvent polarity parameter 1(30) see Section 7.4 for the definition of t(30). 

Optically pure inositol intermediates are very useful for synthesising D-myo-inositol 1,4,5-trisphosphate, which was found to be a second messenger. Ozaki et al [73] studied the resolution of racemic di-O-cylohexylidene-myo-inositol derivatives 17 and 18 by lipase-catalyzed esterification in organic solvents. Lipase from Candida cylindracea exclusively acetylates the hydroxyl group at C-4 or at C-5 of the D-enantiomer of 17 or 18, respectively. Around 100% e.e s of monoacetate products and unreacted starting compounds were obtained. The efficiency of the resolution is affected by the solvent, the most hydrophobic solvents ethyl ether and benzene being more effective than the water miscible solvents such as acetone, THF, and dioxane. 

Reaction mixtures are complex multicomponent systems, and their phase behavior is dictated by the composition of the mixture and operating conditions. Organic solvents present in the reaction medium as reagents may act as cosolvents and result in solute solubility enhancement (as discussed in Section 4.2). For example, the decrease in reaction rate observed at high ethanol concentrations for the lipase-catalyzed esterification of myristic acid + ethanol in SCCO2 has been, in part, attributed to the solubility enhancement of water, resulting in drying of the enzyme... 

Lipase-catalyzed esterification of fatty acids with alcohols [oleic acid + ethanol (141, 152, 163, 169, 178, 195-197), oleic acid + oleyl alcohol (144, 179, 198-200), lauric acid + butanol (142), myristic acid + ethanol (138, 139, 143, 201), stearic acid + ethanol (202), anhydrous milkfat fatty acids + ethanol (197)] in SCCO2 has been widely studied to understand the kinetics/mechanism of the reaction and to determine the effect of operating conditions, substrate concentration, and water content on enzyme activity. Alternative catalysts such as p-toluenesulfonic... 

FIC. 1. Lipase-catalyzed esterification for the production of sugar fatty acid esters in a stirred-tank membrane reactor. 1, pump 2, water bath 3, membrane reactor 4, condenser 5, permeate container 6, vacuum pump. 

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